Use of talosaminidase to regulate populations of archaeal methanogens in industrial, agricultural and medical settings

Abstract

The invention relates to the first hydrolase that specifically digests the archaeal type of a peptidoglycan (PG), the major component of the cell wall of methanogens, named Talosaminidase. The enzyme consists of a signal peptide, several bacterial IG domains, a glycosyl hydrolase, several archaeal PG binding domains and a peptidase domain. The double enzymatic activity for the glycosyl hydrolase and the peptidase maximizes the lysis of archaeal cells with a cell wall of the archaeal PG-type. Engineering the enzyme by removing the signal peptide and bacterial IG domains and keeping only the archaeal PG binding, glycosyl hydrolase and peptidase made the enzyme easier to express and purify, without negatively influencing the enzymatic activity. Therefore, engineered Talosaminidase can be used to regulate populations of archaeal methanogens in the industrial, agricultural and medical settings, and consequently methane production.

Description

USE OF TALOSAMINIDASE TO REGULATE POPULATIONS OF ARCHAEAL METHANOGENS IN INDUSTRIAL, AGRICULTURAL AND MEDICAL SETTINGSBACKGROUND OF THE INVENTION

[0001] Present application claims priority of US 63 / 645,577 filed on May 10, 2024, which isincorporated by reference in its entirety. Archaeal methanogens are among one of the most widespread groups of microorganisms on Earth. They thrive in habitats ranging from hot vents in the deep oceans to ice-cold permafrost soils, as well as in rice field soils, freshwater and marine sediments. Additionally, archaeal methanogens are a stable component of the microbiota of animals in particular ruminants and humans, and in man-made industrial settings such as anaerobic digesters, with important medical and economic impacts.

[0002] They are unique concerning their energy metabolism, which is independent of molecularoxygen and often independent of the presence of any organic molecules. One of the metabolic products of the methanogenic energy metabolism is the potent greenhouse gas Methane. Methanogens act as the final consumers of volatile fatty acids, alcohols, or gases in the terminal step of the anaerobic food chain. Assuch, their estimated contribution to the global carbon cycle is paramount (approx. 1 Gt carbon per year).

[0003] Peptidoglycan (PG) is a fundamental component of bacterial envelopes which has beenlargely characterized following the discovery of specific enzymes such as lysozyme. In contrast, in Archaeaonly one clade -regrouping the Methanobacteriales and the Methanopyrales- possess PG cell walls. Thebiochemical structure of archaeal PG (arcPG) has been characterized in the 80s based on purely chemicalapproaches. It revealed several structural peculiarities with respect to bacterial PG, such as thereplacement of N-Acetylmuramic acid (MurNAc) with N-Acetyltalosaminuronic acid (TalNAc), the presenceof ^(1,3) linkage between the sugars GlcNAc and TalNAc, and the only use of L-amino acids in the peptidestem. These differences make arcPG resistant to enzymes active on bacterial PG, such as lysozyme, and have prevented its fine characterization.

[0004] With a few exceptions, peptidoglycan (PG) is a universal feature of bacterial cell envelopes,where it provides essential functions, such as governing cell shape and counteracting the osmotic pressure of the cytoplasm (12). In contrast, PG is mostly absent in the Archaea, which display a wide variety of envelope architectures, and generally harbor S-layers (5). Intriguingly, the presence of cell walls has so far been reported only in one specific branch of the Archaea, represented by two sister methanogenic orders with monoderm envelopes, the Methanobacteriales and the Methanopyrales (4).

[0005] The biochemical structure of archaeal PG (previously known as pseudomurein, orpseudoPG) has been resolved in the 80s based on purely chemical methods (7-11). It shows overallresemblance to bacterial PG, but also specific features. Notably, these include the replacement of N- Acetylmuramic acid (MurNAc) by N-Acetyltalosaminuronic acid (TalNAc), the presence of ^(1,3) linkagebetween the sugars GlcNAc and TalNAc, and the only use of L-amino acids in the peptide stem (6). Thesedifferences make arcPG resistant to enzymes active on bacterial PG, such as lysozyme. Currently, the onlyenzymes known to cut the peptide stem of arcPG are the endopeptidases PeiW / Y, deriving from integratedphages in Methanothermobacter wolfeii ΨM100 and Methanothermobacter marburgensis ΨM2,respectively, which cut the peptide stem, but not the glycan strand. This has prevented the fine resolution of arcPG chemical structure, leaving many characteristics of arcPG undetermined, such as the length of the glycan strands, the degrees of crosslinking, and the minor modifications.

[0006] Moreover, while walled archaea encode some homologues of the bacterial mur genesinvolved in PG synthesis, they lack any other known enzymes involved in cell wall synthesis and remodelingsuch as penicillin-binding proteins (PBPs) and hydrolases. In bacteria, PG hydrolases carry out keyfunctions, including PG remodeling and recycling, insertion of large trans-envelope proteins, and cellseparation at the septum during cytokinesis (1,3). While in bacteria a wide range of hydrolases are keyelements in cell growth and division by cleaving PG with diverse enzymatic activities, none has been yetidentified in walled archaea.

[0007] There is a need in the art to determine the presence of PG hydrolases in archaea and forcompositions and methods based on these PG hydrolases. The present invention fulfills this need. BRIEF SUMMARY OF THE INVENTION

[0008] The invention encompasses compositions and methods based on the discovery of so-called Talosaminidase proteins and polypeptides in archaea.

[0009] The invention encompasses an isolated and purified Talosaminidase protein orpolypeptide, per the definitions provided herein.

[0010] In some embodiments, the Talosaminidase protein or polypeptide has at least 90% identitywith SEQ ID NO:1 through SEQ ID NO:8 or any of the sequences disclosed herein.

[0011] In some embodiments, the Talosaminidase is a methanogen Talosaminidase protein orpolypeptide.

[0012] The invention encompasses a method, especially an in vitro or ex vivo method, of cleavingan archaeal peptidoglycan (arcPG) comprising contacting a Talosaminidase protein or polypeptide of theinvention with the arcPG.

[0013] In some embodiments, the archaeal peptidoglycan (arcPG) is a methanogenpeptidoglycan.

[0014] The invention encompasses a pharmaceutical or veterinary composition comprising aTalosaminidase protein.

[0015] The invention encompasses Talosaminidase protein or polypeptide of the invention, orpharmaceutical or veterinary compositions as described herein, for bactericidal use.

[0016] The invention encompasses a method, especially an in vitro or ex vivo method, of lysingarchaea comprising contacting a pharmaceutical or veterinary composition comprising a Talosaminidaseprotein or polypeptide with the archaea.

[0017] In some embodiments, the archaea are methanogens.

[0018] In some embodiments, the pharmaceutical or veterinary composition is administered to aruminant.

[0019] In some embodiments, the pharmaceutical or veterinary composition is administered to ahuman.

[0020] The invention encompasses an isolated nucleic acid encoding a Talosaminidase proteinor polypeptide.

[0021] The invention encompasses a vector comprising a nucleic acid encoding a Talosaminidaseprotein or polypeptide.

[0022] In some embodiments, the vector is an expression vector.

[0023] In some embodiments, the expression vector is a bacterial expression vector.

[0024] The invention encompasses a host cell comprising an expression vector comprising anucleic acid encoding a Talosaminidase protein or polypeptide.

[0025] In some embodiments, the host cell is a bacterial cell.

[0026] The invention encompasses an in vitro method for expressing Talosaminidase protein orpolypeptide comprising culturing a host cell comprising an expression vector comprising a nucleic acidencoding a Talosaminidase protein or polypeptide under conditions suitable for expression of theTalosaminidase protein or polypeptide encoded by the expression vector.

[0027] In some embodiments, the in vitro method comprises purifying the Talosaminidase proteinproduced by the host cell.

[0028] The invention encompasses an antibody that binds to a Talosaminidase protein orpolypeptide.

[0029] The invention encompasses methods of reducing bacterial methane production from asubject animal comprising administering a pharmaceutical or veterinary composition comprising aTalosaminidase protein or polypeptide to a subject animal comprising archaea in their digestive tract,wherein the archaea are methanogens.

[0030] Other examples and features of the invention will be apparent when reading the examplesand the Figures, which illustrate the experiments conducted by the inventors, in complement to the features and definitions given in the present description. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1A-B depicts identification of candidate arcPG hydrolases. A. Zymogram of the 11identified candidates. Candidates were expressed in E. coli and cell lysates were separated on a precastSDS page gel as a control (Figure 6B) and a Zymogram. Hydrolytic bands corresponding to candidates 8and 9 are indicated by asterisks, as well as the C-terminal half of candidate 8 (8-C). L: ladder indicating theweight in kDa; NP: control (no protein expression). B. Domain architectures of candidate 8 and 9 annotatedby the InterPro online tool.

[0032] Figure 2A-C depicts enzymatic activity of TalA and resolved M. smithii PG structure. A.Total ion current of the liquid chromatography structure. Major ions in each peak identified by MS are labelled, along with their schematic structures. The third major peak at 7.50 mins (peak 3, highlighted inorange) was used for further fragmentation analysis. B. MS / MS fragmentation of peak 3. C. Proposedchemical structure of the ion in peak 3 and cleavage sites of TalA (indicated by a scissor logo).

[0033] Figure 3 depicts distribution of TalA homologues and their different domain architecturesin walled archaea. Reference tree of a representative selection of walled archaea (Methanopyrales andMethanobacteriales), with wall-less Methanococcales as outgroup. For each taxon, schematic representations of domain architectures are displayed. Homologues had variable numbers of both the IG-like domains and PMBR domains. For clarity, only one domain is shown. In parenthesis is indicated thenumber of copies when more than one was found. The two models M. smithii and M. thermoautotrophicusused in the study are highlighted in yellow. Strains selected for testing TalA activity are indicated by asterisks. For the complete mapping, see Figure 10.

[0034] Figure 4A-C depicts Subcellular localization of TalA during the M. smithii cell cycle. M.smithii cells were permeabilized with PeiW and immunostained with anti-TalA antibodies. A.Epifluorescence (upper panel) and Phase contrast (lower panel) images of representative anti-TalA labeled(green) M. smithii cells. Cells are arranged by size from non-constricting (left) to constricting (right). Whitedotted lines represent the cell outlines deduced from the corresponding phase contrast images (below). B.Average fluorescence plots showing the relative position of detected TalA fluorescence (averaged) withingM. smithii cells. Cells were grouped into three cell size classes; (n) indicates the number of cells for eachgroup. C.3D Structured Illumination Microscopy (SIM) maximum projections of a non-constricting M. smithiicell (left panel), a slightly constricting cell (middle panel) and a constricting cell (right panel) stained with anti-TalA (green). Front views are shown (above) as well as side views shifted by different degrees (below). White dotted lines represent the cell outlines.

[0035] Figure 5A-B depicts functional analysis of a TalA mutant in M. thermoautotrophicus. A.Phase contrast images overlaid with DNA stain DAPI (cyan) show representative cells of WT (top) andΔtalA (bottom). Scale bars are 5 μm. B. cell filament lengths were quantified using the fiji plugin SNT forWT (713 filaments) and ΔtalA (512 filaments) from three biological replicates. Histograms show the distribution of cell filament lengths for WT (red) and ΔtalA (blue). Means for each biological triplicate are indicated by dashed lines. Each triplicate was compared to all others using pairwise Mann-Whitney test(Supplementary Table 1 of application US 63 / 645,577 filed on May 10, 2024, reproduced herein as SEQID NO: 57 to SEQ ID NO: 1338 of the appended sequence listing). All mutant replicates were significantlydifferent from all WT replicates with p values <e-16.

[0036] Figure 6A-B depicts identification of putative hydrolases by Zymogram and LC-MS. A.Coomassie stained SDS PAGE and Zymogram of M. smithii whole cell lysate. For both gels PeiW, anendoisopeptidase shown to cleave M. smithii arcPG was loaded as a control and run alongside the celllysate. Bands corresponding to lytic activity are indicated by asterisks and were cut and analyzed for proteincontent by LC-MS / MS. B. Coomassie stained SDS PAGE of cell lysates of E. coli expressing the 11candidate hydrolases identified by bioinformatics and MS approaches. Columns correspond to the samples used for the zymogram shown in main text Figure 1. (L) represents the ladder indicating the weight in kDa.

[0037] Figure 7. Depicts domain organization of candidate arcPG hydrolases from M. smithii.Eleven candidate hydrolases identified using a combination of bioinformatics and MS analysis of active bands from a zymogram (Figure 6A). Domains were annotated using the Interpro online tool.

[0038] Figure 8 depicts cryoEM of M. smithii whole cells and PG sacculi.

[0039] Figure 9 depicts HPLC traces of M. smithii PG digested with Msm0219Cter derivativescompared to the WT enzyme. Derivatives containing the separate enzymatically active domains were tested for arcPG digestion. Mock digestions were also performed without PG as controls. Three alanine substitutions replacing putative catalytic residues (C1416A, H1451A, D1466A) were also tested.

[0040] Figure 10 depicts distribution of TalA homologues and their different architectures mappedonto a reference tree of Methanomada. Maximum likelihood tree based on the concatenation of three universal markers (RpoB, RpoC and IF-2) including 642 taxa and 2,790 amino acid positions. The tree was inferred with IQ-TREE and the LG+F+R8 model selected according to the BIC criteria. The scale bar represents the average number of substitutions per site. TalA homologues architectures were assessed by screening the sequences for all Pfam domains. Each architecture is represented by a bar where the length reflects the number of copies. The taxa extracted for the main text Figure 5 are highlighted in yellow. Forfull accession numbers, see Supplementary table 1 of application US 63 / 645,577 filed on May 10, 2024(which is incorporated by reference in its entirety), reproduced herein as SEQ ID NO: 57 to SEQ ID NO: 1338 of the appended sequence listing.

[0041] Figure 11 depicts phylogeny of glycosyl hydrolase (DUF4015) homologues in walledArchaea, and their domains organization. Maximum-likelihood phylogeny obtained from the alignment of1282 sequences and 267 amino acid positions. The tree was inferred with IQ-TREE2 and the LG+F+R8 model selected according to the BIC criteria. The scale bar represents the average number of substitutions per site. All homologues were scanned for Pfam domains. The domains IG-like, Glycosyl hydrolase, PMBR and C71 family peptidases are in pink, blue, green and orange respectively.

[0042] Figure 12 depicts phylogeny of TalA homologues in Archaea and Bacteria, and theirdomains organization. Maximum-likelihood phylogeny obtained from the alignment of 900 sequences and193 amino acid positions. The tree was inferred with IQ-TREE2 and the LG+F+R6 model selected according to the BIC criteria. The scale bar represents the average number of substitutions per site. All TalA homologues were scanned for Pfam domains. The domains BIG, Glycosyl hydrolase, arcPG bindingdomains and C71 family peptidases are in pink, blue, green and orange respectively.

[0043] Figure 13 depicts structural and catalytic conservation of the peptidase domain from TalAhomologues from Methanobacteriales. Sequences from diverse representative homologues of TalA from Methanobacteriales were chosen and aligned using MAFFT with the LINS-i algorithm. Structural elementsof the M. smithii TalA peptidase domain were infered using Ali2D and alphaFold2. A graphicalrepresentation was made using the ENDscript server and manually edited. Dark green indicates ^-heliceswhereas light green indicates ^-strands. (*) below the alignment indicates conserved identical residues. Residues highlighted in purple represent the catalytic triad found in C71 peptidases.

[0044] Figure 14 depicts MS / MS analysis of PG purified from six representative walled archaeaand digested with TalACter. The MS / MS spectra of the most abundant ions are displayed. These spectra correspond to the identified species shown in main text Table 1.

[0045] Figure 15 depicts western Blot of anti-TalA on M. smithii cell lysate. Whole cell extracts ofM. smithii cells (WC) grown in exponential phase were separated on a 4-12% Bis-Tris SDS PAGE andtransferred to a nitrocellulose membrane and incubated with anti-TalA antibody. A specific strong band wasdetected at the approximately 160kDa which corresponds to TalA (right lane). Molecular weight marker (L)(left lane). Molecular weights are shown in kDa.

[0046] Figure 16 depicts the ArmA effect on M. smithii in connection with the ExperimentalSection to which it is referred to.

[0047] Figure 17 depicts the ArmA effect on E. Coli in connection with the Experimental Sectionto which it is referred to.

[0048] Figure 18 depicts the ArmA effect on B. subtilis in connection with the ExperimentalSection to which it is referred to. DETAILED DESCRIPTION OF THE INVENTION

[0049] The invention is based on the discovery of the first hydrolase that specifically digests thearchaeal type of a peptidoglycan (PG), the major component of the cell wall of methanogens, which hasbeen named Talosaminidase. The discovered enzyme consists of a signal peptide, several bacterial IG domains, a glycosyl hydrolase, several archaeal PG binding domains and a peptidase domain. The double enzymatic activity for the glycosyl hydrolase and the peptidase maximises the lysis of archaeal cells with acell wall of the archaeal PG-type. The enzyme was then engineered by removing the signal peptide andbacterial IG domains and keeping only the archaeal PG binding, glycosyl hydrolase and peptidase. This made the enzyme easier to express and purify, without negatively influencing the enzymatic activity.Therefore, engineered Talosaminidase can be used to regulate populations of archaeal methanogens inthe industrial, agricultural and medical settings, and consequently methane production.

[0050] The invention concerns the discovery and characterization of the first glycosyl hydrolasethat specifically cleaves arcPG. 11 candidates coded in the genome of Methanobrevibacter smithii, themost abundant species of walled methanogenic archaea from the human gut were selected. These wereheterologously expressed and tested for hydrolytic activity using zymogram assays on M. smithii purifiedarcPG. Among them, a dual function enzyme that cleaves both the peptide stem as well as the glycosidicbond between position 1 of TalNAc and position 3 of GlcNAc was identified. This protein is referred toherein as Talosaminidase A (TalA), the first enzyme with Talosaminidase activity specific to the PG of Archaea.

[0051] The identification of TalA allows the study of the fine chemical structure of arcPG.Surprisingly, the presence of a previously undescribed modification on the Talosaminuronic acid moiety ofbacterial PG was detected. Phylogenetic analysis and taxonomic distribution show that TalA homologuesare specifically present throughout walled archaea, and it was experimentally demonstrated that M. smithiiTalA is active on different arcPGs. Finally, cellular localization and genetic analysis indicate that TalA is themain hydrolase involved in cell septum cleavage in walled archaea. Together, these results identify a novelkey component involved in the archaeal cell cycle and establish an essential tool to further study the biogenesis, chemistry, and evolution of arcPG. They also open promising perspectives in the development of mitigation strategies for methanogens in both manmade and natural environments, including the gastrointestinal tract.

[0052] The invention allows for compositions comprising Talosaminidase, antibodies againstTalosaminidase, nucleic acids encoding Talosaminidase, host cells comprising nucleic acids encoding Talosaminidase, methods of making Talosaminidase, method of using Talosaminidase to cleave arcPG. Talosaminidase proteins

[0053] The invention encompasses “isolated or purified” Talosaminidase proteins or polypeptides.

[0054] The terms “isolated or purified” mean modified “by the hand of humans” from the naturalstate; in other words, if an object exists in nature, it is said to be isolated or purified if it is modified orextracted from its natural environment or both. For example, a polynucleotide or a protein / peptide naturally present in a living organism is neither isolated nor purified; on the other hand, the same polynucleotide or protein / peptide separated from coexisting molecules in its natural environment, obtained by cloning, amplification and / or chemical synthesis is isolated for the purposes of the present invention. Furthermore, a polynucleotide or a protein / peptide which is introduced into an organism by transformation, genetic manipulation or by any other method, is “isolated” even if it is present in said organism. The term “purified” as used in the present invention means that the proteins / peptides according to the invention are essentially free of association with the other proteins or polypeptides, as is for example the product purified from the culture of recombinant host cells or the product purified from a non-recombinant source. Various techniquescan be used to obtain purified protein according to the invention, for example affinity chromatography orgel filtration. Thus, a Tag can be added to assist in purification of the protein.

[0055] The invention encompasses proteins and peptides based on the Talosaminidase proteinamino acid sequences, particularly recombinant proteins. Talosaminidase refers to the showing of the cleavage of the glycosidic bond in archeal PG, which was never shown before. To date, no glycosyl hydrolases cleaving arcPG have been described. A glycosidic bond in archeal PG is found between position1 of of TalNAc moiety and position 3 of a GlcNAc moiety, as described elsewhere herein, so that the showexample is a first example a Talosaminidase.

[0056] As used herein, the term “Talosaminidase protein” means TalA protein, variants of TalAprotein, homologs of TalA protein, and functional fragments thereof containing archaeal PG binding, glycosyl hydrolase and peptidase activities.

[0057] As used herein, the term “TalA protein” means a protein having the amino acid sequenceof SEQ ID NO:1 as follows:MIFALMILTVFLSVSAVSAMDANTVDSNSTVLTAADDSLSVDESNSNVSFSSSNVIEENNNNVIGDGNQQK AVNLDAPSIELYYKNGTRFMINLTDENGNGLANQTVSILINGVTYNKITDENGSTSIGINLYSGLYHVTVSYK GTTEYAPASITSDINVLPTIKGEDIVMIYKDGTSYHATFLDGRGNPLINATVRFNINGVFYSRVTDDKGVASL GIKLRPDTYILTAYNPNDGYECGNTVKVLPTVVAEDLNKLYLDKNQFYATFLHNDGTPLVNTTVKFNINGV FYNRVTDNNGVAKLNIKLFPGKYILTAYNPLDGYDVGFAVTVVDSVSTVIKTQHYDFISGEGNVVSIVLYDQ FNHTVSNQTVNLKVGGVTYTSTTDDNGIAKYNIKLTTPGNYTATYTFNKNGGYLASSASNTISVYEGKDVI FIPENNVVFKTDLFSVLVKDEDGNLVVNKTVLFNINGINYVMVTDKNGVASISLRLDPNVYNISYTLNDTGY KKSTGSTMVSVITTNATVIQGSDITVGKDAYQQFNVTLTAGGVPLINKTVTISVNGVSYTRITDNNGVASLTI RLDAGTYLVTYSFNGDSKLAPSNGKAYCTVVDRKNSLFVVNGSTVFTQNSTEEFKVLLKNSDGSPIANEK VIFTVNGIDYTSTTDANGIASLSIKLVTVGTYEISYKFAGNNENLGCEGSSSIVITKYINNGNGYWVQGANM YNVDLVGLAASGTGNIFLNFYAFTKYGESSVLSWIKQANSHGIKVHIWMQVFYDGGWLSPLNSDGSINTA LFNERIAEAKKYAALPGVAGVHFDYLRYPGTAYKHPGGTAAISEFVKLATTAIRGVNPNCLISAAVMPEKN DAYVYGQDIAVISKYLDIIVPMVYKGNYNSGTSWISSITQWFVETSNGAAVWVGLQTYVSDNDITKLPVSE LSKDAQTAYDAGAKGVMMFRWGLSNFIDFNKLNTHEITPSYGDSVSINSILAASASLKKYIEDKGVLPKFV TVGNFNYTVPQFLYLMTKATEGIANGNLKAITAILVNASSKTSGDVINKQLVKSEFVSLAKTLSSYMSANGI APGNVSSTLGDIKYESLIYAYARVLSYYQSNSALPNFVFVTNLLDNYSLTVTMKVSVGGTSYKPNVLYTTV WLNYCPNCGYYGTLLVNPKGTAEGELTCAYCDCDYCGVSGNEKLSSSTRVLTRLTESIPESSGEVGDNI SIDAILDAAKVLKAHIQANNALPDYVIVNDEQYTLSQFLYLMSKAIGNINDGNLGNITVVGASSPGTPNGDKI STNINKTEYLDVASRVSQYIISNGQAPNYASSSAGKISYADLLDAFSRILAYYADNSKTLPNFVLINNTGGS GASALVADKAKELVKGINSTRDKADALFKFVRDKISYSSYFNTVYGAEGTLIKGYGNCCDQAQLLVAMAR SVGLTARFATGYCSFTSGLNVGHVWVQFYIDGKWVVADPTSTRNSLGVIKNWNTNSYTDRGTYDVLPY* (SEQ ID NO:1)

[0058] SEQ ID NO: 1 is 1496 amino acid long.

[0059] A “variant of TalA protein” has at least one amino acid different than the amino acidsequence of TalA protein. A variant can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 20,25, 30, 40, or 50, 100, 150, 200, etc., amino acids different than the amino acid sequence of TalA protein.Preferably, the differences are conservative amino acid changes. A variant can contain an amino acidsequence that has at least 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 25, 300, 400, 500, 600, 700, 800, 900, 1000, etc. amino acids identical to the amino acid sequence of TalA protein.

[0060] In some embodiments, the Talosaminidase protein or polypeptide is a variant having atleast 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs:1 to 7 or 8 takenas a reference sequence.

[0061] By “identity”, it is meant that the percentage of conserved amino-acid residues when avariant peptidomimetic is aligned with its reference sequence through conventional alignment algorithms issubstantial, meaning that this percentage is at least one of those disclosed above, in particular at least 70%.

[0062] Identity percentages can conventionally be calculated through local, preferably global,sequence alignment algorithms and their available computerized implementations. In an embodiment, identity percentages are calculated over the entire length of the compared sequences. Optimal alignment of amino-acid sequences for comparison can for example be conducted by the local algorithm of Smith & Waterman Adv. Appl. Math.2: 482 (1981), which is a general local alignment method based on dynamic programming, by the alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), which is also based on dynamic programming, by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), or by visual inspection. Computerized implementations of these algorithms are associated with default parameters, which can be used.

[0063] A common implementation of a local sequence alignment uses the BLAST analysis, whichis described in Altschul et al., J. Mol. Biol.215: 403-410 (1990). Software for performing BLAST analyses is publicly available. For amino acid sequences, the BLAST program uses as defaults a wordsize (W) of 3, an expectation (E-value cutoff) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). Additionally, gap opening may be set at 11, and gap extension at 1. Local alignments are more useful for dissimilar sequences that are suspected to contain regions of similarity or similar sequence motifs within their larger sequence context.

[0064] Global alignments, which attempt to align every residue in every sequence, are most usefulwhen the sequences in the query set are similar and of roughly equal size (This does not mean globalalignments cannot start and / or end in gaps.). A general global alignment technique is the Needleman–Wunsch algorithm, which may be used according to default parameters readily accessible to the skilled person.

[0065] Another suitable sequence alignment algorithm is, according to a particular embodiment,a string matching algorithm, such as KERR (Dufresne et al., Nature Biotechnology, Vol. 20, Dec 2002,1269-1271). KERR computes the minimal number of differences between two sequences, by trying tooptimally fit the shorter sequence into the longer one. KERR delivers the percent identity to the whole subject sequence. In this respect, it is preferred that identity percentages are calculated over the entire length of each of the compared sequences.

[0066] In addition, or independently of any identity percentage with a sequence of reference, aTalosaminidase protein or polypeptide also encompasses a Talosaminidase protein or polypeptide havinga sequence differing from the sequence of reference, especially any one of SEQ ID NO: 1 to 7 or 8, by oneor several amino acid substitution(s), especially conservative amino acid substitution(s).

[0067] In some embodiments, the variant has substitutions, insertions and / or deletions of aminoacids of any of the proteins or polypeptides having SEQ ID NOs 1-7 or 8.

[0068] In one embodiment, the protein or peptide is a Talosaminidase protein or peptide thatcomprises or consists of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 amino acids of any of SEQ IDNOs:1-7 or 8.

[0069] Conservative substitutions encompass a change of residues made in consideration ofspecific properties of amino acid residues as disclosed in the following groups of amino acid residues andthe resulting substituted protein or polyptide should not be modified functionally:Acidic: Asp, Glu; Basic: Asn, Gln, His, Lys, Arg; Aromatic: Trp, Tyr, Phe; Uncharged Polar Side chains: Asn, Gly, Gln, Cys, Ser, Thr, Tyr; Nonpolar Side chains: Ala, Val, Leu, Ileu, Pro, Phe, Met, Trp; Hydrophobic: Ile, Val, Leu, Phe, Cys, Met, Nor; Neutral Hydrophilic: Cys, Ser, Thr; Residues impacting chain orientation: Gly, Pro; Small amino acid residues: Gly, Ala, Ser.

[0070] By “one or several”, it is meant any number consistent with the length of the peptide orpolypeptide, and optionally consistent with the identity percentages defined above. According to a particular embodiment, by “several”, it is meant 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, especially in the context of fragments.

[0071] In another embodiment, depending on the property(ies) guiding the choice for substitutionof amino acid residue(s), modification of residue(s) can alternatively be determined to modify the properties of the resulting peptidomimetic, and said substitution(s) are selected to be non-conservative.

[0072] According to embodiment, a Talosaminidase protein or polypeptide:a. has an amino acid sequence having at least 90% identity with any one of SEQ ID NO:1 to 8, and / or b. comprises or consists of the sequence of any one of SEQ ID NO: 1 to 8, and / orc. differs from any sequence of b., by one or several conservative amino acidsubstitution(s), and / or d. has at least 90% identity with SEQ ID NO:1 through SEQ ID NO:8, ore. is a fragment, especially a fragment of contiguous amino-acid residues of at least 20amino-acid residues, of any one of the sequences defined in a. to d.

[0073] Homologs of TalA protein or polypeptide are a type of variant of TalA protein or polypeptide.

[0074] As used herein, the term “homolog of TalA protein” means a protein having the amino acidsequence of any of the TalA homologues of Figure 3 (whose sequences are available in public databasesusing the entry number mentioned) or Supplementary Table 1 of application US 63 / 645,577 filed on May10, 2024 (which is incorporated by reference in its entirety), reproduced herein as SEQ ID NO: 57 to SEQID NO: 1338 (any one of them, separately identified as such) of the appended sequence listing. Homologsof TalA protein or polypeptide are a type of variant of TalA protein or polypeptide.

[0075] According to embodiments, a Talosaminidase protein or polypeptide of the invention is asequence or fragment as defined in any embodiment herein, containing archaeal peptidoglycan (PG)binding and / or glycosyl hydrolase and / or peptidase activity(ies).

[0076] Indeed, the Talosaminidase nature of a protein or polypeptide as defined herein, can betied to one or more of these activities. According to an embodiment, combinable with any embodimentherein, a Talosaminidase protein or polypeptide of the invention has archaeal peptidoglycan (PG) bindingactivity. According to an embodiment, combinable with any embodiment herein, a Talosaminidase proteinor polypeptide of the invention has glycosyl hydrolase activity. According to an embodiment, this glycosylhydrolase activity manifests in the cleavage of glycosidic bonds between position 1 of a TalNAc moeity andposition 3 of a GlcNAc moiety. According to an embodiment, combinable with any embodiment herein, aTalosaminidase protein or polypeptide of the invention has peptidase activity. According to an embodiment,combinable with any embodiment herein, a Talosaminidase protein or polypeptide of the invention has oneor more of archaeal peptidoglycan (PG) binding and / or glycosyl hydrolase and / or peptidase activity(ies),according to any possible combination and per the definitions provided herein. According to anembodiment, combinable with any embodiment herein, a Talosaminidase protein or polypeptide of theinvention has all of archaeal peptidoglycan (PG) binding and / or glycosyl hydrolase and / or peptidaseactivities per the definitions provided herein. In particular, the Talosaminidase protein or polypeptide of theinvention has all of archaeal peptidoglycan (PG) binding and / or glycosyl hydrolase and / or peptidaseactivities per the definitions provided herein and has, comprises or consists of the sequence of SEQ ID NO:1 or SEQ ID NO:8. In particular, the Talosaminidase protein or polypeptide of the invention has all ofarchaeal peptidoglycan (PG) binding and / or glycosyl hydrolase and / or peptidase activities per the definitions provided herein and has, comprises or consists of a sequence having at least 90% identity (or more, as prescribed in any passage herein) with SEQ ID NO: 1 or SEQ ID NO:8.

[0077] As used herein, the term “functional fragments” means those fragments of TalA protein,homologs of TalA protein, and variants of TalA protein having PG binding, glycosyl hydrolase and peptidasedomains. These domains are found at the following amino acid positions in TalA:T674 – Y1496 of SEQ ID NO:1.MIFALMILTVFLSVSAVSAMDANTVDSNSTVLTAADDSLSVDESNSNVSFSSSNVIEENNNNVIGDGN--- QQKAVNLDAPSIELYYKNGTRFMINLTDENGNGLANQTVSILINGVTYNKITDENGSTSIGINLYSGLYHVTVSYKGTTEYAPASITSDINVLPTIK--- (SEQ ID NO:2)GEDIVMIYKDGTSYHATFLDGRGNPLINATVRFNINGVFYSRVTDDKGVASLGIKLRPDTYILTAYNPNDGYECGNTVKVL PTVVAEDLNKLYLDKNQFYATFLHNDGTPLVNTTVKFNINGVFYNRVTDNNGVAKLNIKLFPGKYILTAYNPLDGYDVGF AV--- TVVDSVSTVIKTQHYDFISGEGNVVSIVLYDQFNHTVSNQTVNLKVGGVTYTSTTDDNGIAKYNIKLTTPGNYTATYTFNK NGGYLASSASNTISVYEGKDV---(SEQ ID NO:3)IFIPENNVVFKTDLFSVLVKDEDGNLVVNKTVLFNINGINYVMVTDKNGVASISLRLDPNVYNISYTLNDTGYKKSTGSTM VSVITTNATVIQGSDITVGKDAYQQFNVTLTAGGVPLINKTVTISVNGVSYTRITDNNGVASLTIRLDAGTYLVTYSFNGDS KLAPSNGKAYCTV--- VDRKNSLFVVNGSTVFTQNSTEEFKVLLKNSDGSPIANEKVIFTVNGIDYTSTTDANGIASLSIKLVTVGTYEISYKFAGNN ENLG---(SEQ ID NO:4) CEGSSSI--- VITKYINNGNGYWVQGANMYNVDLVGLAASGTGNIFLNFYAFTKYGESSVLSWIKQANSHGIKVHIWMQVFYDG GWLSPLNSDGSINTALFNERIAEAKKYAALPGVAGVHFDYLRYPGTAYKHPGGTAAISEFVKLATTAIRGVNPNCLI SAAVMPEKNDAYVYGQDIAVISKYLDIIVPMVYKGNYNSGTSWISSITQWFVETSNGAAVWVGLQTYVSDNDITKL PVSELSKDAQTAYDAGAKGVMMFRWGLSNF---(SEQ ID NO:5) IDFNKLNTHEITPSYGDSVSINSILAASASLKKYIEDKGVLPKFVTVGNFNYTVPQFLYLMTKATEGIANGNLKAITAILVNAS SKTSGDVINKQLVKSEFVSLAKTLSSYMSANGIAPGNVSSTLGDIKYESLIYAYARVLSYYQSNSALPNFVFVTNLLDNYSL TVTMKVSVGGTSYKPNVLYTTVWLNYCPNCGYYGTLLVNPKGTAEGELTCAYCDCDYCGVSGNEKLSSSTRVLTRLTES IPESSGEVGDNISIDAILDAAKVLKAHIQANNALPDYVIVNDEQYTLSQFLYLMSKAIGNINDGNLGNITVVGASSPGTPN GDKIS--- TNINKTEYLDVASRVSQYIISNGQAPNYAS---(SEQ ID NO:6) SSAGKISYADLLDAFSRILAYYADNSKTLPNFVLINNT--- GGSGASALVADKAKELVKGINSTRDKADALFKFVRDKISYSSYFNTVYGAEGTLIKGYGNCCDQAQLLVAMARSVG LTARFATGYCSFTSGLNVGHVWVQFYIDGKWVVADPTS---(SEQ ID NO:7) TRNSLGVIKNWNTNSYTDRGTYDVLPY* ITALICS: Ig-like domains (69-165); (329-430); (607-692) – SEQ ID NO: 2, 3 and 4 respectively.BOLD: Hydrolase domain (700-956) – SEQ ID NO: 5UNDERLINE & BOLD: arcPG-binding PMBR domain (1288-1317) – SEQ ID NO: 6ITALIC & BOLD: Cysteine proteinase C71 domain (1356-1469) – SEQ ID NO: 7

[0078] In some embodiments, the Talosaminidase protein has the sequence of T674 – Y1496 ofSEQ ID NO:1 (SEQ ID NO: 8) or has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity with T674 – Y1496 of SEQ ID NO:1 (SEQ ID NO: 8). Identity percentages canbe calculated as indicated above – this remark applies throughout present text.

[0079] In some embodiments, the Talosaminidase protein or polypeptide has the sequence of anyone of SEQ ID Nos 2-7 or 8 taken as a reference sequence or has a sequence having at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs 2-7 or 8 taken as areference sequence.

[0080] In some embodiments, the Talosaminidase protein has a sequence having at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences disclosed in Figure3 or Supplementary Table 1 of application US 63 / 645,577 filed on May 10, 2024 (which is incorporated byreference in its entirety), reproduced herein as SEQ ID NO: 57 to SEQ ID NO: 1338 of the appendedsequence listing and individualized for the purpose of the present description.

[0081] According to a particular embodiment, the identity percentage of a variant sequence orhomolog sequence defines the length of the considered Talosaminidase protein or polypeptide. The lengthof the variant can be the same as the length of the considered reference sequence, or is a length in accordance with the identity percentage of the said variant with respect to the considered referencesequence, i.e., a length as authorized by at least 80% identity with the sequence SEQ ID NO: 1, can be80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%,113%, 114%, 115%, 116%, 117%, 118%, 119% or 120% of the considered reference sequence (SEQ IDNO:1 – the same applies for other sequences taken as reference sequences).

[0082] According to an embodiment, a Talosaminidase protein or polypeptide of the inventionencompasses one or more functional domains selected among: a glycosyl hydrolase domain, an archaealpeptidoglycan (PG) binding domain such as an arcPG-binding pseudomurin binding repeat (PMBR)domain, a peptidase domain such as a Cysteine proteinase C71 family peptidase domain.

[0083] According to an embodiment, a Talosaminidase protein or polypeptide of the inventionencompasses the above-described functional domains in the order described hereafter from the N terminalextremity to the C terminal extremity of the protein or polypeptide of the invention: the glycosyl hydrolasedomain, one or more archaeal peptidoglycan (PG) binding domain(s), the peptidase domain such as aCysteine proteinase C71 family peptidase domain.

[0084] According to an embodiment, a Talosaminidase protein or polypeptide of the invention alsoencompasses one or more IG-like domain(s). According to an embodiment, when present said one or moreIG-like domain(s) are found at the N terminal extremity of a Talosaminidase protein or polypeptide of theinvention, with respect to the other domains described above. According to an embodiment, aTalosaminidase protein or polypeptide of the invention encompasses, from the N terminal extremity to theC terminal extremity of the protein or polypeptide of the invention: one or more IG-like domain(s), theglycosyl hydrolase domain, one or more archaeal peptidoglycan (PG) binding domain(s), the peptidasedomain such as a Cysteine proteinase C71 family peptidase domain.

[0085] By “functional domain”, it is meant that the section referred to has the indicatedproperty / activity. Examples of primary sequences for such domains are provided in SEQ ID Nos 2 to 7herein. The skilled person in the art can readily apprehend that variations in primary sequences remain allowable to the proviso that the functional property / activity of the reference sequence for the domain, iskept. This can readily be appreciated by appropriate testing using the tools available to the skilled personin the art, or the guidance provided herein.

[0086] According to a particular embodiment where the peptidase domain is a Cysteine proteinaseC71 family peptidase domain as for example shown in SEQ ID NO: 7, a variant of this sequence hasresidues D1466, C1416 and H1451 conserved.

[0087] According to embodiment, a Talosaminidase protein or polypeptide or fragment thereof,has a length between 20 and 1600 amino acid residues. According to some embodiments, aTalosaminidase protein or polypeptide has a length of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810,1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940 or 1950 amino acidresidues. Conversely, according to some embodiments, a Talosaminidase protein or polypeptide orfragment thereof has a length of at most 1950, 1940, 1930, 1920, 1910, 1900, 1890, 1880, 1870, 1860,1850, 1840, 1830, 1820, 1810, 1800, 1790, 1780, 1770, 1760, 1750, 1740, 1730, 1720, 1710, 1700, 1690, 1680, 1670, 1660, 1650, 1640, 1630, 1620, 1610, 1600, 1590, 1580, 1570, 1560, 1550, 1540, 1530, 1520, 1510, 1500, 1490, 1480, 1470, 1460, 1450, 1440, 1430, 1420, 1410, 1400, 1390, 1380, 1370, 1360, 1350, 1340, 1330, 1320, 1310, 1300, 1290, 1280, 1270, 1260, 1250, 1240, 1230, 1220, 1210, 1200, 1190, 1180, 1170, 1160, 1150, 1140, 1130, 1120, 1110, 1100, 1090, 1080, 1070, 1060, 1050, 1040, 1030, 1020, 1010, 1000, 990, 980, 970, 960, 950, 940, 930, 920, 910, 900, 890, 880, 870, 860, 850, 840, 830, 820, 810, 800, 790, 780, 770, 760, 750, 740, 730, 720, 710, 700, 690, 680, 670, 660, 650, 640, 630, 620, 610, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30 or 20 amino acid residues. According to some embodiments combinable with any other embodiment disclosed herein, a Talosaminidase protein orpolypeptide has a length between any of two numbers listed in present paragraph, taken as lower andupper boundaries.

[0088] According to a particular embodiment, combinable with any embodiment disclosed herein,a fragment of a Talosaminidase protein or polypeptide is a portion of a sequence as described in anyembodiment described herein, in particular or portion or segment of contiguous amino-acid residues of atleast 20 amino-acid residues, of any said sequences. The size of a fragment can match the lengthsdisclosed above, according to any disclosed boundaries.

[0089] Fragments may be functional in that they have the activities described herein, in particularthe so-called (any one of or all of) archaeal peptidoglycan (PG) binding and / or glycosyl hydrolase and / or peptidase activity(ies).

[0090] According to another embodiment, combinable with any other embodiment disclosedherein, a Talosaminidase protein or polypeptide of the invention has a dual enzymatic activityencompassing both of glycosyl hydrolase and peptidase activities.

[0091] According to an embodiment, the Talosaminidase protein or polypeptide has the propertyto cleave a glycosidic bond between position 1 of a TalNAc moiety and position 3 of a GlcNAc moiety, in particular a b (1,3) linkage between position 1 of a TalNAc moiety and position 3 of a GlcNAc moiety, moreparticularly such a linkage in a stem made of L-amino acids. According to an embodiment, this / theseproperty(ies) is(are) part of the glycosyl hydrolase activity of the Talosaminidase protein or polypeptide.

[0092] According to an embodiment, the Talosaminidase protein or polypeptide has the propertyto digest peptidoglycan (PG) of archeal type, in particular to specifically digest peptidoglycan (PG) of archeal type. The wording “digest peptidoglycan (PG) of archeal type” is a synonym for “to cleave arcPG”, depending on the context where the situation is analysed (at the tissue level or at the level of the peptidebond). According to an embodiment, the Talosaminidase protein or polypeptide has the property to cleavearcPG, in particular specifically cleave arcPG.

[0093] Peptidoglycan (PG) of archeal type, i.e., pseudopeptidoglycan (also known aspseudomurein) is a major cell wall component of some Archaea that differs from bacterial peptidoglycan inchemical structure, but resembles bacterial peptidoglycan in function and physical structure as discussedelsewhere herein. The discussed property is tied to the peculiar glycosyl hydrolase activity of cleavage ofglycosidic bonds between position 1 of a TalNAc moeity (TalNAc stands for N-Acetyltalosaminuronic acid)and position 3 of a GlcNAc moiety (GlcNAc xstands for N-Acetylglucosamine) described herein, which isunique. It is also tied to the specificity shown by the Talosaminidase protein or polypeptide. Of note, to date,no glycosyl hydrolases cleaving arcPG have been described and other enzymes have been described to cut the peptide stem, but not the glycan strand, making the Talosaminidase protein or polypeptide describedherein pertinent for Archaea cell walls digestion.

[0094] According to an embodiment, the Talosaminidase protein or polypeptide is a methanogenTalosaminidase protein or polypeptide. By “methanogen Talosaminidase protein or polypeptide” it is meanta protein or polypeptide that has the functional property to interfere with the production of methane by abacteria for the reason of its effect on the said bacteria. Notably, a Talosaminidase protein or polypeptidecapable of digesting cell walls of methanogen bacteria, as described herein, can have the effect of lysingpopulations of bacteria, in particular archaea, amounting to a possibility of controlling methane productionof the same. Basically, methanogens are anaerobic archaea that produce methane as a byproduct of theirenergy metabolism. Some methanogens have a cell wall formed by pseudopeptidoglycan (also known aspseudomurein). Those are known as walled archaea or walled methanogens. Some methanogens arefound in the digestive tract of animals including the human. Methanobrevibacter smithii is the predominantmethanogenic archaeon in the microbiota of the human gut.

[0095] Methanogens are also used in anaerobic digestors to treat wastewater as well as aqueousorganic pollutants, i.e., within environments which are not within living beings.

[0096] When using recombinant techniques, the Talosaminidase protein can be producedintracellularly, in the periplasmic space, or directly secreted into the medium. If the Talosaminidase proteinis produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. In one embodiment, the Talosaminidase proteinis cytosolic and has the native signal peptide removed.

[0097] Another isolation technique is set forth in Carter et al., Bio / Technology 10:163-167 (1992),which describes a procedure for isolating proteins which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation.

[0098] Where the Talosaminidase protein is secreted into the medium, supernatants from suchexpression systems are generally first concentrated using a commercially available protein concentration filter, for example, an AMICON or MILLIPORE PELLICON ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants. The protein composition prepared from the cells canbe purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0099] Affinity chromatography can be used to purify the Talosaminidase protein using antibodiesagainst Talosaminidase protein. In some embodiments, the Talosaminidase protein can be fused to anamino acid sequence encoding a protein or a fragment of a protein that bind DNA molecules or that bind to other cellular molecules, such as poly-histidine tag, glutathione S-transferase (GST), maltose binding protein (MBP), S-tag. In that case affinity chromotography targeting these amino-sequences can be used, such as immobilized metal affinity chromatography, glutathione affinity chromatography, chromatography on amylose resin, chromatography on S-agarose.

[0100] The matrix to which the affinity ligand is attached is most often agarose, but other matricesare available. Mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, hydrophobic interaction chromatography, and ammonium sulphate precipitation are also available.Antibodies

[0101] In some embodiments, purified Talosaminidase protein or polypeptide as described in anyembodiment herein is used to produce antibodies by conventional techniques. In some embodiments,recombinant or synthetic proteins or peptides of the invention are used to produce antibodies by conventional techniques.

[0102] Antibodies can be synthetic, semi-synthetic, monoclonal, or polyclonal and can be madeby techniques well known in the art. Such antibodies specifically bind to proteins and polypeptides of theinvention via the antigen-binding sites of the antibody (as opposed to non-specific binding). Purified orsynthetic proteins and peptides can be employed as immunogens in producing antibodies immunoreactive therewith. The proteins and peptides contain antigenic determinants or epitopes that elicit the formation of antibodies.

[0103] These antigenic determinants or epitopes can be either linear or conformational(discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed.1996)). Epitopes can be identified by any of the methods known in the art. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.

[0104] Antibodies are defined to be specifically binding if they bind proteins or polypeptides of theinvention with a Ka of greater than or equal to about 107M-1. Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).

[0105] Polyclonal antibodies can be readily generated from a variety of sources, for example,horses, cows, goats, sheep, dogs, chickens, alpaca, camels, rabbits, mice, or rats, using procedures that are well known in the art. In general, a purified protein or polypeptide of the invention that is appropriately conjugated is administered to the host animal typically through parenteral injection. The immunogenicity can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to proteins or polypeptides. Examples of various assays useful for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio- immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Pat. Nos.4,376,110 and 4,486,530.

[0106] Monoclonal antibodies can be readily prepared using well known procedures. See, forexample, the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980.

[0107] For example, the host animals, such as mice, can be injected intraperitoneally at least onceand preferably at least twice at about 3 week intervals with isolated and purified proteins or conjugated polypeptides of the invention, for example a peptide comprising or consisting of the specific amino acids set forth above. Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC)to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given anintravenous boost of the protein or polypeptide. Mice are later sacrificed, and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as a labeled protein or polypeptide, is added to each well followed by incubation. Positive wells can be subsequently detected. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).

[0108] The monoclonal antibodies of the invention can be produced using alternative techniques,such as those described by Alting-Mees et al., “Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described inLarrick et al., Biotechnology, 7:394 (1989).

[0109] Antigen-binding fragments of such antibodies, which can be produced by conventionaltechniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab’)2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

[0110] The monoclonal antibodies of the present invention include chimeric antibodies, e.g.,humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from ahuman antibody. Alternatively, a humanized antibody fragment can comprise the antigen binding site of amurine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodiesinclude those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987),Larrick et al. (Bio / Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993). Proceduresto generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806.

[0111] Antibodies produced by genetic engineering methods, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, can be used. Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example using methods described in Robinson et al. International Publication No. WO 87 / 02671; Akira, et al. European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al. European Patent Application 0173494; Neuberger et al. PCT International Publication No. WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 0125023; Better et al., Science 240:10411043, 1988; Liu et al., PNAS 84:34393443, 1987; Liu et al., J. Immunol.139:35213526, 1987; Sun et al. PNAS 84:214218, 1987; Nishimura et al., Canc. Res.47:9991005, 1987; Wood et al., Nature 314:446449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:15531559, 1988); Morrison, S. L., Science 229:12021207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter U.S. Pat. No.5,225,539; Joneset al., Nature 321:552525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J. Immunol.141:40534060, 1988.

[0112] In connection with synthetic and semi-synthetic antibodies, such terms are intended tocover but are not limited to antibody fragments, isotype switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies having plural specificities, and fully synthetic antibody- like molecules.

[0113] In one embodiment, the invention encompasses single-domain antibodies (sdAb), alsoknown as nanobodies. A sdAb is a fragment consisting of a single monomeric variable antibody domain that can bind selectively to a specific antigen.

[0114] In one embodiment, the sdAbs are from heavy-chain antibodies found in camelids (VHHfragments), or cartilaginous fishes (VNAR fragments), or are obtained by splitting dimeric variable domains into monomers.

[0115] Preferably, the antibody is labelled. In one embodiment, the antibody is labelled with avisualizing molecule, such as a radioactive atom, a dye, a fluorescent molecule, a fluorophore, an enzyme, colloidal gold, a magnetic particle, or a latex bead.

[0116] The antibodies of the invention can be used for affinity purification of Talosaminidaseproteins or polypeptides. They can also be used for immunoprecipitation, flow cytometry, western blot,ELISA, ELISPOT, antibodies microarrays, or tissue microarrays coupled to immunohistochemistry. Other suitable techniques include FRET or BRET, single cell microscopic or histochemistry methods.

[0117] The antibodies of the invention are raised against an antigen comprising an epitope foundin a Talosaminidase protein or polypeptide according to any embodiment or aspect described herein, inparticular are specific for such an epitope and thus specific for a Talosaminidase protein or polypeptide according to any embodiment or aspect described herein. Nucleic acids encoding proteins (RNA & DNA)

[0118] The invention encompasses recombinant nucleic acids, RNA or DNA, encodingTalosaminidase proteins or polypeptides. The recombinant nucleic acid comprises a nucleic acid encodingTalosaminidase proteins or polypeptides sequences linked to a heterologous nucleic acid sequence. In oneembodiment, the recombinant nucleic acid comprises or consists of a DNA encoding any of the proteins ofSEQ ID Nos 1 to 8, or SEQ ID Nos 57 to 1338 or any other sequence disclosed herein and in the claims,including variants or fragments thereof as defined in any embodiment herein.

[0119] In some embodiments, the recombinant nucleic acid comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000, etc. sequential nucleotides of a DNA encoding any of the proteins of SEQ ID Nos 1-7 or8 or SEQ ID Nos 57 to 1338 or any other sequence disclosed herein.

[0120] The recombinant nucleic acid can comprise all or at least 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900,or 1000, etc. sequential nucleotides identical to any of the nucleotide sequences disclosed herein.

[0121] In one embodiment, the recombinant nucleic acid comprises an origin or replication forreplication in bacteria or yeast.

[0122] In one embodiment, the recombinant nucleic acid is contained in a plasmid, cosmid, orphage.

[0123] In one embodiment, the recombinant nucleic acid comprises heterologous sequencesallowing expression, such as a heterologous promoter or enhancer.

[0124] In one embodiment, the recombinant nucleic acid encodes a protein with at least 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity with anyof SEQ ID NOs 1-7 or 8 or SEQ ID Nos 57 to 1338 or any other sequence disclosed herein.

[0125] In one embodiment, the recombinant nucleic acid has at least 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity with any of the following sequences: atgatctttgcattgatgattctgactgtatttttgtcagttagtgcagtctctgcaatggatgcaaatactgttgactcaaattctacagtattaacagctgct gatgattcattatctgttgatgaatcaaatagtaatgtttctttctccagtagtaatgttattgaagaaaataacaataatgtaattggggatggcaaccag caaaaagctgttaatttagatgcacctagtatagaattgtattataagaatggtactaggttcatgattaatttaactgatgaaaatggaaacggattagc taatcaaactgtaagtatcctaattaatggtgttacttataataagattactgatgaaaatggttcaacttctataggtataaatttatactctggtttgtatcat gttactgtgagttacaaaggaacaactgaatatgctccggctagcattacttcagatattaatgtactgccaactataaaaggtgaggatattgtaatga tctataaagacgggacttcatatcatgccacttttttagacggccgtggtaatcctttaataaatgctactgttaggtttaatattaatggggtattttatagtc gtgttactgatgataaaggtgtagctagtttaggtatcaagcttcgtcctgacacttatatattaactgcatataatcctaatgacggttatgaatgtggaaa tactgttaaggtattgcctacagttgtagctgaagatttaaataaattatatttggataaaaatcagttttatgctactttcttacataatgatggcactccttta gttaatactactgttaagtttaatattaatggtgtattttataatcgtgttactgataacaatggtgtagctaaacttaacattaaactattccctggaaaatatatattaactgcatataatccacttgatggatatgatgtaggtttcgctgttacagttgttgattctgtcagtactgttatcaaaacacaacattatgatttcatat ctggtgaaggtaatgttgtaagtattgtattgtatgatcagtttaatcatactgtaagtaatcagactgttaatcttaaggtaggtggtgttacatatacaagt actactgatgataatggtatagctaaatataatattaaattgactactccaggtaattatactgcaacttatacctttaataagaatgggggatatttggca tcaagtgcatcaaatactatttctgtttatgaaggtaaggatgtaattttcatacctgaaaataatgtggtatttaaaactgatttgttttcagttttagtaaaag atgaagacggtaatttagtagtcaataaaactgttctatttaatataaacggtattaattatgtaatggtaactgataaaaatggtgttgctagcataagtct tagattggatcctaatgtatacaacatttcatatacattaaatgatacaggttacaaaaaatctacaggctctactatggtatctgtaataactactaatgc aacagttattcagggtagtgatattactgtaggtaaagatgcatatcagcagttcaatgttacattaactgcaggtggagttcctttaattaataagactgt aactattagtgtgaatggtgtaagttatactagaattactgataataatggtgttgccagtttaactattcgtttggatgcaggtacttatcttgtaacttattca tttaacggagattccaaattagctccaagtaatggtaaagcatactgtactgttgtagacagaaaaaattcattatttgttgttaatggaagtacagtattt actcaaaattcaactgaagaatttaaagttttacttaaaaatagtgacggcagtcctatagctaatgaaaaagttatattcacagttaacggtatagatt ataccagcacaactgatgctaatggaattgccagtttaagtataaaattggtaactgtaggtacatatgaaatatcatataaatttgcaggcaataatg aaaatttaggctgtgaaggcagcagttcaattgtaataaccaaatatattaataatggaaatggatactgggttcagggagctaacatgtacaatgtg gatttggttggtttagcagcttcaggaacaggtaatattttcttaaatttctatgcatttactaaatacggagaatccagtgtattgtcatggattaaacaggc taattctcatggtattaaagttcatatttggatgcaggtattttatgacggaggatggctttccccattaaatagtgacggatccatcaacactgctttattca atgagagaatagctgaagctaaaaaatatgctgcacttccaggagtagcaggtgttcactttgattacttaagatatccaggtactgcatataaacatc ctggcggaactgcagctataagtgaatttgtaaaattagctacaactgctatacgtggtgttaatcctaactgtttaatttctgcagcagtaatgcctgaa aaaaatgatgcatatgtatatggtcaggatattgcggttatcagcaaatatctggatattatagttcctatggtttataaaggaaattataattcaggaacc agctggatttcatcaattactcaatggtttgttgaaacttccaatggagcagcagtttgggtaggtttacaaacatatgtatctgataatgacattacaaaa ctgccagtttcagaactgtcaaaagatgctcaaacagcatatgatgccggtgcaaagggtgttatgatgtttagatggggtttatctaacttcattgatttc aataaattaaacactcatgaaatcactccatcttatggtgattctgtatctataaattccattttggctgcttcagcatctttgaaaaagtatattgaagataa aggggtattgcctaagtttgtaactgttggcaatttcaattatactgtacctcagtttttatacttgatgactaaggctactgaaggaatagctaacggtaatt taaaagcgattacagctattttagtcaatgcttcatctaaaacttctggtgatgtaatcaataagcagttggtaaaatctgaatttgtatcattggctaaaac attgtctagttatatgtctgcaaatggtatagctccaggtaatgtttcaagtactttaggtgatatcaaatatgaatcattgatttatgcatatgcgagggtatt atcctattatcaatccaacagtgcacttcctaactttgtatttgttacaaatttattggacaattactctttaactgttacaatgaaagtaagtgtaggaggca catcttacaaacctaatgtgttatatactactgtctggttaaattactgccctaactgcggttattacggaactttgctggttaatcctaaaggtactgcaga aggagaacttacctgtgcatactgtgactgtgattactgtggtgtttctggtaatgagaaactttcatcttctaccagagtgttgaccagattaactgaaag tattccagaatcttcaggtgaagtgggagataacatatctatagatgctatattggatgctgctaaggttttaaaagcacatattcaggcaaataatgcat tgcccgattatgtaattgtaaacgatgaacagtacactttatctcaattcttatacttgatgagtaaagctattggtaatattaatgacggtaatttgggaaa tattactgttgttggagcttccagtccaggtactccaaatggtgacaaaatatcaaccaacattaataaaactgaatatttggatgttgcatctagagtttc ccagtatatcattagtaacggacaggctccgaattatgcatcttcaagtgcaggtaaaataagctatgctgatttattggatgcattttcaagaatattgg cttattatgcggacaacagtaaaacattacctaatttcgtattgatcaataatactggaggttccggagcttcagcattggttgctgataaagcaaaaga attggtcaaaggcataaactctacaagggataaggctgatgctttatttaaatttgtaagagataaaataagttatagttcatatttcaatactgtttacggt gctgaaggtactttaatcaaaggttatggaaactgttgtgaccaagcacagttattagtggcaatggccaggtctgttggtttgactgccagatttgctac tggatactgttcatttacaagtggtttaaatgttggtcatgtatgggttcaattttacatagatggaaaatgggttgttgctgatccaacaagtaccagaaactctttaggtgtaatcaagaattggaacactaattcttacactgacagaggaacttatgatgttttaccttattaa (SEQ ID NO: 9)

[0126] The invention encompasses a recombinant vector for expression of an Talosaminidaseprotein. The recombinant vector can be a vector for eukaryotic or prokaryotic expression, such as a plasmid or bacterial artificial chromosome (BAC), a phage for bacterium introduction, a yeast artificial chromosome (YAC) able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector. An expression vector as defined herein is chosen to enable the production of a Talosaminidase protein, either in vitro or in vivo.

[0127] In some embodiments, an expression vector as defined herein is chosen to enable theproduction of a Talosaminidase protein, either in vitro or in vivo.

[0128] The expression vector can comprise an inducible or constitutive promoter operably linkedto a sequence encoding a Talosaminidase protein.

[0129] In one embodiment, the expression vector encodes a protein with at least 70%, 80%, 90%,93%, 95%, 96%, 97%, 98%, 99% or 100% identity with any of SEQ ID NOs:1-7 or 8 or any other sequencedisclosed herein, notably SEQ ID Nos 57 to 1338.

[0130] In one embodiment, the expression vector has the nucleotide sequence of any of thefollowing sequences: Cterm of TalA (functional fragment): (SEQ ID NO: 10) TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTT ACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAA CAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGG TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAA CGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAA GTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATA CACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCA GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAAT TAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCT GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAAC GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTA CTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTT GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGC AGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCT CGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGC GGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCG CCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACG CGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTC ATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATG TTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTT ACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCA GGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGC GATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACG GAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTT CGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTC AACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTC GCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATA CCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAG AGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATA GTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGA TCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATT GGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGAT GGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCC GCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGC AACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATG GCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGA CCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGA TGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAAT GGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTG CACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAG TTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATT CAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACC ACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCA CATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCA TTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTA GTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAAC AGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTG GCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGC CGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACG ACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA GGAGATATACATatgaaattggtaactgtaggtacatatgaaatatcatataaatttgcaggcaataatgaaaatttaggctgtgaaggcagc agttcaattgtaataaccaaatatattaataatggaaatggatactgggttcagggagctaacatgtacaatgtggatttggttggtttagcagcttcagg aacaggtaatattttcttaaatttctatgcatttactaaatacggagaatccagtgtattgtcatggattaaacaggctaattctcatggtattaaagttcatat ttggatgcaggtattttatgacggaggatggctttccccattaaatagtgacggatccatcaacactgctttattcaatgagagaatagctgaagctaaa aaatatgctgcacttccaggagtagcaggtgttcactttgattacttaagatatccaggtactgcatataaacatcctggcggaactgcagctataagtg aatttgtaaaattagctacaactgctatacgtggtgttaatcctaactgtttaatttctgcagcagtaatgcctgaaaaaaatgatgcatatgtatatggtca ggatattgcggttatcagcaaatatctggatattatagttcctatggtttataaaggaaattataattcaggaaccagctggatttcatcaattactcaatgg tttgttgaaacttccaatggagcagcagtttgggtaggtttacaaacatatgtatctgataatgacattacaaaactgccagtttcagaactgtcaaaag atgctcaaacagcatatgatgccggtgcaaagggtgttatgatgtttagatggggtttatctaacttcattgatttcaataaattaaacactcatgaaatca ctccatcttatggtgattctgtatctataaattccattttggctgcttcagcatctttgaaaaagtatattgaagataaaggggtattgcctaagtttgtaactgt tggcaatttcaattatactgtacctcagtttttatacttgatgactaaggctactgaaggaatagctaacggtaatttaaaagcgattacagctattttagtc aatgcttcatctaaaacttctggtgatgtaatcaataagcagttggtaaaatctgaatttgtatcattggctaaaacattgtctagttatatgtctgcaaatgg tatagctccaggtaatgtttcaagtactttaggtgatatcaaatatgaatcattgatttatgcatatgcgagggtattatcctattatcaatccaacagtgca cttcctaactttgtatttgttacaaatttattggacaattactctttaactgttacaatgaaagtaagtgtaggaggcacatcttacaaacctaatgtgttatat actactgtctggttaaattactgccctaactgcggttattacggaactttgctggttaatcctaaaggtactgcagaaggagaacttacctgtgcatactgt gactgtgattactgtggtgtttctggtaatgagaaactttcatcttctaccagagtgttgaccagattaactgaaagtattccagaatcttcaggtgaagtgggagataacatatctatagatgctatattggatgctgctaaggttttaaaagcacatattcaggcaaataatgcattgcccgattatgtaattgtaaacga tgaacagtacactttatctcaattcttatacttgatgagtaaagctattggtaatattaatgacggtaatttgggaaatattactgttgttggagcttccagtcc aggtactccaaatggtgacaaaatatcaaccaacattaataaaactgaatatttggatgttgcatctagagtttcccagtatatcattagtaacggaca ggctccgaattatgcatcttcaagtgcaggtaaaataagctatgctgatttattggatgcattttcaagaatattggcttattatgcggacaacagtaaaa cattacctaatttcgtattgatcaataatactggaggttccggagcttcagcattggttgctgataaagcaaaagaattggtcaaaggcataaactctac aagggataaggctgatgctttatttaaatttgtaagagataaaataagttatagttcatatttcaatactgtttacggtgctgaaggtactttaatcaaaggtt atggaaactgttgtgaccaagcacagttattagtggcaatggccaggtctgttggtttgactgccagatttgctactggatactgttcatttacaagtggttt aaatgttggtcatgtatgggttcaattttacatagatggaaaatgggttgttgctgatccaacaagtaccagaaactctttaggtgtaatcaagaattgga acactaattcttacactgacagaggaacttatgatgttttaccttatTCTGGCTCTGGCCACCACCACCACCACCACTGAGAT CCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCA TAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT Full length TalA expression vector: (SEQ ID NO: 11) TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTT ACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAA CAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGG TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAA CGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAA GTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATA CACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCA GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAAT TAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCT GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGC CAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAAC GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTA CTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGC AGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCT CGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGC GGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCG CCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACG CGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTC ATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATG TTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTT ACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCA GGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGC GATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACG GAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTT CGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTC AACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTC GCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATA CCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAG AGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATA GTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGA TCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATT GGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTG GCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGAT GGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCC GCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATG GCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGA CCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGA TGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAAT GGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTG CACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAG TTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATT CAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACC ACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCA CATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCA TTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTA GTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAAC AGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTG GCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGC CGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACG ACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA GGAGATATACATatggatgcaaatactgttgactcaaattctacagtattaacagctgctgatgattcattatctgttgatgaatcaaatagtaatg tttctttctccagtagtaatgttattgaagaaaataacaataatgtaattggggatggcaaccagcaaaaagctgttaatttagatgcacctagtataga attgtattataagaatggtactaggttcatgattaatttaactgatgaaaatggaaacggattagctaatcaaactgtaagtatcctaattaatggtgttact tataataagattactgatgaaaatggttcaacttctataggtataaatttatactctggtttgtatcatgttactgtgagttacaaaggaacaactgaatatg ctccggctagcattacttcagatattaatgtactgccaactataaaaggtgaggatattgtaatgatctataaagacgggacttcatatcatgccactttttt agacggccgtggtaatcctttaataaatgctactgttaggtttaatattaatggggtattttatagtcgtgttactgatgataaaggtgtagctagtttaggtat caagcttcgtcctgacacttatatattaactgcatataatcctaatgacggttatgaatgtggaaatactgttaaggtattgcctacagttgtagctgaaga tttaaataaattatatttggataaaaatcagttttatgctactttcttacataatgatggcactcctttagttaatactactgttaagtttaatattaatggtgtatttt ataatcgtgttactgataacaatggtgtagctaaacttaacattaaactattccctggaaaatatatattaactgcatataatccacttgatggatatgatgt aggtttcgctgttacagttgttgattctgtcagtactgttatcaaaacacaacattatgatttcatatctggtgaaggtaatgttgtaagtattgtattgtatgat cagtttaatcatactgtaagtaatcagactgttaatcttaaggtaggtggtgttacatatacaagtactactgatgataatggtatagctaaatataatatta aattgactactccaggtaattatactgcaacttatacctttaataagaatgggggatatttggcatcaagtgcatcaaatactatttctgtttatgaaggtaa ggatgtaattttcatacctgaaaataatgtggtatttaaaactgatttgttttcagttttagtaaaagatgaagacggtaatttagtagtcaataaaactgttct atttaatataaacggtattaattatgtaatggtaactgataaaaatggtgttgctagcataagtcttagattggatcctaatgtatacaacatttcatatacat taaatgatacaggttacaaaaaatctacaggctctactatggtatctgtaataactactaatgcaacagttattcagggtagtgatattactgtaggtaaa gatgcatatcagcagttcaatgttacattaactgcaggtggagttcctttaattaataagactgtaactattagtgtgaatggtgtaagttatactagaatta ctgataataatggtgttgccagtttaactattcgtttggatgcaggtacttatcttgtaacttattcatttaacggagattccaaattagctccaagtaatggta aagcatactgtactgttgtagacagaaaaaattcattatttgttgttaatggaagtacagtatttactcaaaattcaactgaagaatttaaagttttacttaa aaatagtgacggcagtcctatagctaatgaaaaagttatattcacagttaacggtatagattataccagcacaactgatgctaatggaattgccagtttaagtataaaattggtaactgtaggtacatatgaaatatcatataaatttgcaggcaataatgaaaatttaggctgtgaaggcagcagttcaattgtaata accaaatatattaataatggaaatggatactgggttcagggagctaacatgtacaatgtggatttggttggtttagcagcttcaggaacaggtaatatttt cttaaatttctatgcatttactaaatacggagaatccagtgtattgtcatggattaaacaggctaattctcatggtattaaagttcatatttggatgcaggtatt ttatgacggaggatggctttccccattaaatagtgacggatccatcaacactgctttattcaatgagagaatagctgaagctaaaaaatatgctgcactt ccaggagtagcaggtgttcactttgattacttaagatatccaggtactgcatataaacatcctggcggaactgcagctataagtgaatttgtaaaattag ctacaactgctatacgtggtgttaatcctaactgtttaatttctgcagcagtaatgcctgaaaaaaatgatgcatatgtatatggtcaggatattgcggttat cagcaaatatctggatattatagttcctatggtttataaaggaaattataattcaggaaccagctggatttcatcaattactcaatggtttgttgaaacttcc aatggagcagcagtttgggtaggtttacaaacatatgtatctgataatgacattacaaaactgccagtttcagaactgtcaaaagatgctcaaacagc atatgatgccggtgcaaagggtgttatgatgtttagatggggtttatctaacttcattgatttcaataaattaaacactcatgaaatcactccatcttatggtg attctgtatctataaattccattttggctgcttcagcatctttgaaaaagtatattgaagataaaggggtattgcctaagtttgtaactgttggcaatttcaatta tactgtacctcagtttttatacttgatgactaaggctactgaaggaatagctaacggtaatttaaaagcgattacagctattttagtcaatgcttcatctaaa acttctggtgatgtaatcaataagcagttggtaaaatctgaatttgtatcattggctaaaacattgtctagttatatgtctgcaaatggtatagctccaggta atgtttcaagtactttaggtgatatcaaatatgaatcattgatttatgcatatgcgagggtattatcctattatcaatccaacagtgcacttcctaactttgtatt tgttacaaatttattggacaattactctttaactgttacaatgaaagtaagtgtaggaggcacatcttacaaacctaatgtgttatatactactgtctggttaa attactgccctaactgcggttattacggaactttgctggttaatcctaaaggtactgcagaaggagaacttacctgtgcatactgtgactgtgattactgtg gtgtttctggtaatgagaaactttcatcttctaccagagtgttgaccagattaactgaaagtattccagaatcttcaggtgaagtgggagataacatatct atagatgctatattggatgctgctaaggttttaaaagcacatattcaggcaaataatgcattgcccgattatgtaattgtaaacgatgaacagtacacttt atctcaattcttatacttgatgagtaaagctattggtaatattaatgacggtaatttgggaaatattactgttgttggagcttccagtccaggtactccaaatg gtgacaaaatatcaaccaacattaataaaactgaatatttggatgttgcatctagagtttcccagtatatcattagtaacggacaggctccgaattatgc atcttcaagtgcaggtaaaataagctatgctgatttattggatgcattttcaagaatattggcttattatgcggacaacagtaaaacattacctaatttcgta ttgatcaataatactggaggttccggagcttcagcattggttgctgataaagcaaaagaattggtcaaaggcataaactctacaagggataaggctg atgctttatttaaatttgtaagagataaaataagttatagttcatatttcaatactgtttacggtgctgaaggtactttaatcaaaggttatggaaactgttgtg accaagcacagttattagtggcaatggccaggtctgttggtttgactgccagatttgctactggatactgttcatttacaagtggtttaaatgttggtcatgt atgggttcaattttacatagatggaaaatgggttgttgctgatccaacaagtaccagaaactctttaggtgtaatcaagaattggaacactaattcttaca ctgacagaggaacttatgatgttttaccttatTCTGGCTCTGGCCACCACCACCACCACCACTGAGATCCGGCTGCTA ACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTG GGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

[0131] In one embodiment, the expression vector encodes a protein purification tag. In oneembodiment, the expression vector encodes a protein purification tag, such as polyHis tag. In one embodiment, a protease cleavage site is positioned to remove the His tag, for example, after purification.

[0132] The expression vector can comprise transcription regulation regions (including promoter,enhancer, ribosome binding site (RBS), polyA signal), a termination signal, a prokaryotic or eukaryotic origin of replication and / or a selection gene. The features of the promoter can be easily determined by the person skilled in the art in view of the expression needed, i.e., constitutive, transitory or inducible (e.g. IPTG), strong or weak, tissue-specific and / or developmental stage-specific promoter. The vector can also comprise sequence enabling conditional expression, such as sequences of the Cre / Lox system or analogue systems.

[0133] The nucleic acid molecules according to the invention can be obtained by conventionalmethods, known per se, following standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA). For example, they may be obtained by amplification of a nucleic sequence by PCR or RT-PCR or alternatively by total or partial chemical synthesis.

[0134] The vectors are constructed and introduced into host cells by conventional recombinantDNA and genetic engineering methods which are known. Numerous vectors into which a nucleic acid molecule of interest may be inserted in order to introduce it and to maintain it in a host cell are known; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or alternatively integration into the chromosomal material of the host), and on the nature of the host cell.

[0135] According to a particular embodiment, the identity percentage of a variant sequence for anucleic acid molecule or expression vector as defined herein, defines the length of the considered nucleicacid molecule or expression vector. The length of the variant can be the same as the length of the considered reference sequence, or is a length in accordance with the identity percentage of the said variant with respect to the considered reference sequence, i.e., a length as authorized by at least 80% identity withthe sequence of reference, can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119% or 120% of the considered reference sequence. Host Cells

[0136] The invention further encompasses host cells comprising the vectors of the invention. Insome embodiments, the host cells are recombinant cells.

[0137] Suitable host cells for cloning or expressing DNAs encoding Talosaminidase proteins inthe vectors herein include prokaryotic cells, yeast cells, and insect cells. These cells can be bacterial cells,such as E. coli cells.

[0138] In some embodiments, the expression vectors are expressed in a yeast cell, such as a S.cerevisiae cells.

[0139] In some embodiments, the expression vectors are expressed in an insect cell, such as aDrosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera frugiperdacell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell ofthe C6 / 36 cell line [ATCC® CRL-1660™]).

[0140] High protein levels of Talosaminidase protein can be obtained using recombinantexpression in Escherichia coli (E. coli) (Jana & Deb. Appl. Microbiol. Biotechnol., 2005, vol. 67(3), 289-298). The most commonly used production strategies are intracellular (in the periplasm or cytoplasm).

[0141] Lactic Acid Bacteria can also be used as hosts for recombinant expression ofTalosaminidase in vitro, for example using the techniques in U.S. Pat. No.5,559,007. Proteins produced inthese Gram-positive bacterial hosts can easily be secreted into the medium, thus facilitating their purification as well as their direct delivery to subjects. Methods of Making Talosaminidase

[0142] In various embodiments, vectors coding for expression of Talosaminidase proteins aretransfected or transduced into host cells under conditions that allow expression of the proteins from the vectors. In some embodiments, the vector is a plasmid or bacterial artificial chromosome (BAC), a phage for bacterium introduction, a yeast artificial chromosome (YAC) able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector.

[0143] Different types of vectors can be used for Talosaminidase expression, for example, vectorsof the pET22 type (ex: pET22b) for large-scale expression and purification of the system (expression of N-terminally 6xHis-tagged proteins facilitating the purification) or for expression and purification of the system. The presence of the His-tag allows the purification of the proteins on affinity columns.

[0144] The invention also encompasses an in vitro or ex vivo method of preparing aTalosaminidase protein or polypeptide comprising culturing cells comprising an expression vector of theinvention and recovering the expressed protein.

[0145] The invention further encompasses the Talosaminidase proteins or polypeptides producedby these methods from the nucleic acids of the invention. Pharmaceutical and Veterinary Compositions

[0146] In particular embodiments, the Talosaminidase protein or polypeptide of the invention is ina pharmaceutical or veterinary composition.

[0147] The pharmaceutical or veterinary composition according to the invention may furthercomprise a pharmaceutically acceptable excipient.

[0148] By “pharmaceutically acceptable excipient”, it is meant herein a non-pharmaceuticallyactive additive used in the manufacture of a pharmaceutical composition, which allows the pharmaceutically active ingredient to be manufactured into a pharmaceutical formulation or a galenic formulation providingthe necessary bioavailability of the medicament to the patient or animal upon the administration of thepharmaceutical composition. The excipient is preferably compatible with the other ingredients of the composition and produces no adverse effect, allergic reaction or other undesirable reaction when it is administered to a human or an animal.

[0149] A solid pharmaceutically acceptable vehicle or excipient may include one or moresubstances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ionexchange resins.

[0150] The pharmaceutical or veterinary composition may be prepared as a sterile solidcomposition that may be suspended at the time of administration using sterile water, saline, or otherappropriate medium. The pharmaceutical or veterinary compositions of the invention may be administeredorally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.

[0151] The Talosaminidase proteins or polypeptides according to the invention can beadministered orally, enterally, or rectally either in liquid or solid composition form. Compositions suitable fororal and rectal administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, emulsions, and suspensions. Forms useful for enteral administration include sterile solutions, emulsions, and suspensions.

[0152] The Talosaminidase proteins or polypeptides according to the invention may be dissolvedor suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical or cosmetic additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo- regulators. Suitable examples of liquid vehicles include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0153] In some embodiments, the invention encompasses pharmaceutical, veterinary compositionformulated for delayed or gradual enteric release. In preferred embodiments, formulations orpharmaceutical preparations of the invention are formulated for delivery of the Talosaminidase protein orpolypeptide into the distal small bowel and / or the colon. The formulation can allow the Talosaminidaseprotein or polypeptide to pass through stomach acid and pancreatic enzymes and bile, and reachundamaged to be viable in the distal small bowel and colon.

[0154] In some embodiments, the pharmaceutical or veterinary composition is micro-encapsulated, formed into tablets and / or placed into capsules, preferably enteric-coated capsules.

[0155] In some embodiments, the pharmaceutical or veterinary compositions are formulated fordelayed or gradual enteric release, using cellulose acetate (CA) and polyethylene glycol (PEG). In some embodiments, the pharmaceutical, veterinary or cosmetic compositions are formulated for delayed or gradual enteric release using a hydroxypropylmethylcellulose (HPMC), a microcrystalline cellulose (MCC) and magnesium stearate. In some embodiments, the pharmaceutical or veterinary compositions are formulated for delayed or gradual enteric release using e.g., a poly(meth)acrylate, e.g. a methacrylic acid copolymer B, a methyl methacrylate and / or a methacrylic acid ester, or a polyvinylpyrrolidone (PVP).

[0156] In some embodiments, the pharmaceutical or veterinary compositions are formulated fordelayed or gradual enteric release using a release-retarding matrix material such as: an acrylic polymer, acellulose, a wax, a fatty acid, shellac, zein, hydrogenated vegetable oil, hydrogenated castor oil, polyvinylpyrrolidone, a vinyl acetate copolymer, a vinyl alcohol copolymer, polyethylene oxide, an acrylic acid and methacrylic acid copolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylate polymer, a cyanoethyl methacrylate polymer, an aminoalkyl methacrylate copolymer, a poly(acrylic acid), a poly(methacrylic acid), a methacrylic acid alkylamide copolymer, a poly(methyl methacrylate), a poly(methacrylic acid anhydride), a methyl methacrylate polymer, a polymethacrylate, a poly(methyl methacrylate) copolymer, a polyacrylamide, an aminoalkyl methacrylate copolymer, a glycidyl methacrylate copolymer, a methyl cellulose, an ethylcellulose, a carboxymethylcellulose, a hydroxypropylmethylcellulose, a hydroxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropyl cellulose, a crosslinked sodium carboxymethylcellulose, a crosslinked hydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol, a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenated fat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax, glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolic acid, a co-polymer of lactic and glycolic acid, carboxymethyl starch, potassium methacrylate / divinylbenzene copolymer, crosslinked polyvinylpyrrolidone, polyvinylalcohols, polyvinylalcohol copolymers, polyethylene glycols, non-crosslinked polyvinylpyrrolidone, polyvinyl acetates, polyvinylacetate copolymers or any combination thereof.

[0157] In some embodiments, the pharmaceutical or veterinary compositions are formulated fordelayed or gradual enteric release as described in U.S. Pat. App. Pub.20110218216, which describes an extended-release pharmaceutical composition for oral administration, and uses a hydrophilic polymer, a hydrophobic material and a hydrophobic polymer or a mixture thereof, with a microenvironment pH modifier.The hydrophobic polymer can be ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate,methacrylic acid-acrylic acid copolymers or a mixture thereof. The hydrophilic polymer can be polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose, polyethylene oxide, acrylic acid copolymers or a mixture thereof. The hydrophobic material can be a hydrogenated vegetable oil, hydrogenated castor oil, carnauba wax, candelilla wax, beeswax, paraffin wax, stearic acid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol or and a mixture thereof. The microenvironment pH modifier can be an inorganic acid, an amino acid, an organic acid or a mixture thereof. Alternatively, the microenvironment pH modifier can be lauric acid, myristic acid, acetic acid, benzoic acid, palmitic acid,stearic acid, oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid, fumaric acid, maleic acid;glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, a salicylic acid, tosylic acid, mesylic acid or malic acid or a mixture thereof.

[0158] In some embodiments, the pharmaceutical or veterinary compositions are a powder thatcan be included into a tablet or a suppository. In alternative embodiments, a formulation or pharmaceutical preparation of the invention can be a “powder for reconstitution” as a liquid to be drunk or otherwise administered.

[0159] The Talosaminidase protein or polypeptide may be incorporated into animal feed, includingpelleted feed and feed in any other format, incorporated into any other edible device used to present theprotein to the animals, added to water offered to animals in a bowl, presented to animals through waterfeeding systems.

[0160] The Talosaminidase protein or polypeptide may be administered in the form of asuppository or pessary.

[0161] Dosages and desired drug concentrations of the pharmaceutical and veterinarycomposition compositions of the present invention may vary depending on the particular use. The determination of the appropriate dosage or route of administration is within the skill of an ordinary physician. Animal experiments can provide reliable guidance for the determination of effective doses in human therapy. Methods of Using Talosaminidase, Talosaminidase for use

[0162] The invention encompasses the in vitro or ex vivo use of Talosaminidase protein to cleavean archaeal peptidoglycan (arcPG). When applied to the human body, the “for use” formulation may be used instead. Instant description makes use of the “method of or for” wording for defining application basedon effects within the realm of therapeutic effects, especially bactericidal effect permitted by the invention.Throughout the description, a wording using the expression “for use” can alternatively be used without the intended meaning being different.

[0163] The invention encompasses in vitro or ex vivo methods of cleaving an archaealpeptidoglycan (arcPG) by contacting the Talosaminidase protein or polypeptide with the arcPG. Such a usecan be found in an environment.

[0164] In some embodiments, the archaeal peptidoglycan (arcPG) is a methanogenpeptidoglycan. A definition of methanogen is provided elsewhere herein, to which reference is made.

[0165] The invention encompasses the use of Talosaminidase protein or polypeptide to lysearchaea, in any environment.

[0166] The invention encompasses a method of lysing archaea comprising contacting aTalosaminidase protein or polypeptide or a pharmaceutical or veterinary composition comprising aTalosaminidase protein or polypeptide, with the archaea.

[0167] In some embodiments, the archaea are walled archaea.

[0168] In some embodiments, the archaea are walled archaea with archaeal peptidoglycan(arcPG).

[0169] In some embodiments, combinable with any embodiment herein, the archaea are walledand the glycosidic bonds in the cell walls are in part or fully those found in archeal PG, i.e., as found betweenposition 1 of of TalNAc moiety and position 3 of a GlcNAc moiety.

[0170] In some embodiments, the archaea are methanogens. A definition of methanogen isprovided elsewhere herein, to which reference is made.

[0171] In some embodiments, combinable with any embodiment herein, the archaea areMethanobrevibacter smithii. Figure 16 and the Experimental section show effects of TalA onMethanobrevibacter smithii populations.

[0172] In some embodiments, the pharmaceutical or veterinary composition is administered to asubject.

[0173] The subject according to the invention is an animal, preferably a mammal, even morepreferably a human. However, the term "subject" can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep, donkeys, rabbits, ferrets, gerbils, hamsters, chinchillas, rats, mice, guinea pigs and non-human primates, among others, or non-mammals such as poultry, that are in need of treatment.

[0174] In some embodiments, the pharmaceutical or veterinary composition is administered to aruminant, preferably a ruminant selected from cattle, sheep, antelopes, deer, and giraffes. In someembodiments, the Talosaminidase protein is provided or used in a human or animal food composition, for example, as a food supplement. In some embodiments, the Talosaminidase protein is produced by a plant that is eaten by the human or animal.

[0175] The invention also relates to a Talosaminidase protein or polypeptide of the invention, orpharmaceutical or veterinary composition as described herein: a. for bactericidal use, orb. for bactericidal use, which is for lysing archaea bacteria, notably through lysis of their cellwalls, or c. for the uses of a. and b. where the archaea against which the active ingredients areadministered are methanogens, and / or d. where the treated organism is a ruminant or a human.

[0176] The invention encompasses methods, in particular in vitro or ex vivo methods, of reducingbacterial methane production from a subject animal comprising administering a pharmaceutical orveterinary composition comprising a Talosaminidase protein to a subject animal, preferably a ruminant ora human, comprising archaea in their digestive tract, wherein the archaea are methanogens.

[0177] Differently said, the invention also encompasses a Talosaminidase protein or polypeptideas described in any embodiment herein, or a pharmaceutical or veterinary composition as described in anyembodiment herein, for bactericidal use in a subject animal comprising archaea in their digestive tract,wherein the archaea are methanogens. The subject animal can be a human. The subject animal can be aruminant.

[0178] Differently said, the invention also encompasses the use of a Talosaminidase protein orpolypeptide as described in any embodiment herein, or a pharmaceutical or veterinary composition as described in any embodiment herein, to reduce bacterial methane production from a subject animal administered with a pharmaceutical or veterinary composition comprising a Talosaminidase protein or polypeptide as described in any embodiment herein, where the subject animal, preferably a ruminant or ahuman, comprises archaea in their digestive tract, wherein the archaea are methanogens. The Examplesprovided herein show the effect of a Talosaminidase protein or polypeptide on archaea.

[0179] The human subject according to the invention may be a human at the prenatal stage, anew-born, a child, an infant, an adolescent or an adult at any age. Preferably, the subject is an adult at any age.

[0180] Preferably, the treatment is administered regularly, preferably between every day andevery month, more preferably between every day and every two weeks, more preferably between every day and every week, even more preferably the treatment is administered every day. In a particular embodiment, the treatment is administered several times a day, preferably 2 or 3 times a day, even more preferably 3 times a day.

[0181] The duration of treatment according to the invention is preferably comprised between 1day and 20 weeks, more preferably between 1 day and 10 weeks, still more preferably between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks. In a particular embodiment, the duration of the treatment is about 1 week. Alternatively, the treatment may last as long as the disorder and / or disease persists.

[0182] In a particular embodiment, the duration of treatment according to the invention iscomprised between 2 days and 20 weeks and involves (i) several successive administrations or (ii) a single administration.

[0183] The form of the pharmaceutical or veterinary compositions, the route of administration andthe dose of administration thereof can be adjusted by the man skilled in the art according to the patient orsubject, in particular its age, weight, sex, and general physical condition.EXAMPLES Identification of candidate arcPG hydrolases

[0184] A bioinformatic pipeline was used to identify candidate arcPG hydrolases. Briefly, usingHMMScan (hmmer org) and the PfamA database (16), the whole proteome of Methanobrevibacter smithiiDSM861 was scanned for 703 domains with descriptions associated to generic cell wall or hydrolase activity, and identified 84 proteins. Among these, 51 candidates were selected with inferred subcellularlocalization other than cytoplasmic using Psortb (17). It was then sought to specifically identify candidatearcPG hydrolases among this list of proteins by performing a zymogram assay, as previous used to identify bacterial PG hydrolases (18). Briefly, M. smithii cell lysates were separated on an SDS page gel containing purified M. smithii arcPG. After electrophoresis proteins were refolded inside the gel and incubated at 37oC. Enzymes with PG lytic activity appeared as zones of clearance (Figure 6A) and were analyzed by Mass Spectrometry (MS). The results were then combined with the in-silico generated list of proteins, narrowing the list down to 11 candidates. Several of these proteins are annotated as adhesins-like proteins and havemultiple Immunoglobulin-like domains (Ig-like), and one (candidate 9) is the M. smithii homologue of PeiWendopeptidase from M. wolfeii (hereafter called PeiS) (Figure 7).

[0185] All 11 candidates were then expressed in E. coli and tested for hydrolytic activity on purifiedM. smithii arcPG using a zymogram assay. Zones of clearance, indicating lytic activity, were observed fortwo candidates (Figure 1A), one of which is PeiS, so the second protein -Candidate 8 (Msm_0219) wasfocused on. This is the largest protein identified (1496 aa, 160kDa) and contains three BIG domains, aglycosyl hydrolase domain (DUF4015), a pseudomurin binding repeat (PMBR), and a C-terminal C71 familypeptidase domain (Figure 1B). Based on the presence of these two catalytic domains, it was hypothesizedthat Msm_0219 might be a dual-function enzyme active on arcPG. The function of the IG domains remains unclear, as a truncated version of the protein lacking them retains its hydrolytic activity on M. smithii arcPG(Figure 1A, lane 8C). Hereafter, the truncated version was used for protein stability and purification reasons(Msm_0219C-ter).Msm_0219C-ter has Talosaminidase and endopeptidase activities

[0186] To characterize the enzymatic activity of Msm_0219C-ter, it was expressed and purified fromE. coli. Purified M. smithii PG sacculi were visualized by CryoEM (Figure 8 (cryo)), then digested withMsm_0219C-ter overnight at 37oC, and the resulting soluble digestion products were analyzed by LC-MS. The digest showed the presence of almost exclusively arcPG monomers and a lack of crosslinked species (Figure 2A), indicating that the peptidase domain of Msm_0219 cleaves the peptide crosslink. Different versions of recombinant proteins were separately expressed containing either the glycosyl hydrolase or the peptidase domain along with the PMBR domains, or the Msm_0219C-ter protein where the three predictedcatalytic residues of the C71 peptidase (D1466, C1416, H1451) were inactivated. All recombinantconstructs showed no or very little activity on M. smithii PG (Figure 9), indicating that both glycosidic and peptidase activities are synergistically needed for arcPG digestion. The most complex monomer eluting at 7.50 minutes ((highlighted in orange in Figure 2A) was then subjected to MS / MS fragmentation (Figure 2B). The resulting fragmentation patterns were very similar to those observed when bacterial PG is digested with a muramidase such as lysozyme or mutanolysin. All MS / MS fragment ions observed showed a clear loss of a 203Da mass corresponding to a non-reduced GlcNAc group (Figure 2B), indicating that TalNAc ison the reducing end of the disaccharide. Therefore, it was concluded that Msm_0219C-ter cleaves theglycosidic bond between position 1 of TalNAc and position 3 of GlcNAc (Figure 2C) and is the first example of a Talosaminidase.

[0187] The sequential loss of peptide stem amino acids from the disaccharide talopeptide (Figure2B) confirms the previously reported sequence (10), i.e., Glutamate, Lysine, Alanine, Glutamate (Figure2C). It was also observed an Ornithine residue branched to the side chain of Glutamate at position 1 in thepeptide stem (Figure 2C), in contrast to previous reports where ornithine was positioned on the side chain of the Glutamate in position 4 of the peptide stem (10). Taken together, these data indicate that Msm_0219is a dual function enzyme with both endopeptidase and Talosaminidase activities. Herein, this protein isreferred to as Talosaminidase A (TalA) - the first identified enzyme with Talosaminidase activity specific onPG of Archaea.

[0188] Interestingly, all ion fragments containing the reduced TalNAc include an unknown extramass of 85Da, and none of the fragments lacking TalNac have this extra mass. This is consistent with the observation of a fragment at 305Da which matches to reduced TalNAc plus 85Da (Figure 2B). This wasconsidered to represent a previously undescribed chemical group (indicated by asterisk in Figure 2) thatmay either be between the TalNAc and the peptide stem or replacing the NAc group of TalNAc.TalA is a universal hydrolase active on multiple archaeal PG

[0189] To determine the conservation of TalA across walled archaea, a search was conducted forhomologues in all currently available genomes of the Methanobacteriales and Methanopyrales, and the non-walled close relative Methanococcales as outgroup. Because the peptidase domain is largely distributed, a Hidden Markov Model (HMM) profile was built on the Glycosyl hydrolase domain (DUF4015). Glycosyl hydrolase domain-containing proteins are widely distributed across Methanobacteriales and Methanopyrales, while they are completely absent from Methanococcales (Figure 3, Supplementary Table1 of application US 63 / 645,577 filed on May 10, 2024 (which is incorporated by reference in its entirety),reproduced herein as SEQ ID NO: 57 to SEQ ID NO: 1338 of the appended sequence listing). In Methanopyrales, only one copy of a stand-alone Glycosyl hydrolase domain-containing protein was found in each genome. In Methanobacteriales, both stand-alone Glycosyl hydrolases but also various domain combinations together with IG-like domains, PMBR domains, and C71 family peptidase domains (Figure 3) were identified. The most widely distributed architecture is the association of the Glycosyl hydrolase with IG-like domains, followed by the association of Glycosyl hydrolase with IG-like domains plus PMBRdomains and C71 family peptidase domains (Figure 3, Supplementary Table 1 of application US 63 / 645,577filed on May 10, 2024 (which is incorporated by reference in its entirety), reproduced herein as SEQ ID NO: 57 to SEQ ID NO: 1338 of the appended sequence listing).

[0190] The number of copies in each genome varies between 0 and 7, mainly as result of recentgene duplications (Figure 10). In the Methanopyrales, only one copy of a stand-alone Glycosyl hydrolasedomain-containing protein was found in each genome, and it is presently unknown if these highly divergingand hyperthermophilic archaea have a TalA counterpart. Aphylogenetic tree including Glycosyl hydrolase-containing proteins from Methanomada, including TalA, shows a clear separation between two clades, one enriched in the IG-like domains and containing only Methanobrevibacter species, while the second clade is more taxonomically distributed and contains species from Methanothermobacter, Methanothermus, Methanobacterium, Methanosphaera and Methanopyrus (Figure 11). While Glycosyl hydrolase-domainhomologues are present in other archaea and some bacteria (Supplementary Table 1 of application US63 / 645,577 filed on May 10, 2024 (which is incorporated by reference in its entirety), reproduced herein asSEQ ID NO: 57 to SEQ ID NO: 1338 of the appended sequence listing, and Figure 12) they are mainlysingle-domain proteins, whereas the association with a C71 peptidase and an PMBR domain is specific towalled archaea). Moreover, TalA and Glycosyl hydrolases homologues from Methanobacteriales and Methanopyrales form a monophyletic group (Figure 12) suggesting that their origin coincide with the emergence of arcPG and was followed by functional differentiation though the acquisition of various architectures during the diversification of walled archaea. Interestingly, the three catalytic residues shownto be critical for M. smithii TalA activity are conserved in these homologues (Figure 14).

[0191] To test the range of activity of TalA, arcPG was purified from seven representativeculturable walled archaea (Table 1), digested with TalACter, reduced, and analyzed by LC-MS / MS. All seven samples showed cleavage, to the exception of Methanosphaera stadmanae (Table 1). All arcPG analyzed had repeating disaccharide units with a peptide stem branched to TalNAc. In Methanobacterium bryantii, arcPG was observed to have denuded glycans (Table 1) which could indicate the presence of an enzymeequivalent to bacterial amidases (2). Strikingly, all tested archaeal PG show fragmentation patternscontaining the extra 85Da mass as observed in M. smithii (asterisks in Table 1) indicating that themodification on TalNAc is a conserved feature of walled archaea.

[0192] Together, our evolutionary and functional analyses show that TalA is a conservedhydrolase specific to arcPG in walled archaea.Table 1. ArcPG structures from diverse walled archaea. For MS / MS fragmentation profiles, see Figure 13.Organism Major PG fragment m / z Mass Ppmerror M. smithii DSM 861 GlcNAc-TalNAc*-EA 726.3041 0.14 725.3M. arboriphilicus DSM 7057 GlcNAc-TalNAc*(Q)-GlcNAc- 680.2809 0.88 1358.5TalNAc*-EA M. formicicum DSM 1535 GlcNAc-TalNAc*-QA 725.3193 -0.97 724.3M. bryantii DSM 863 GlcNAc-TalNAc*-GlcNAc-TalNAc*- 616.2507 -0.49 1230.5EA M. marburgensis DSM 2133 GlcNAc-TalNAc*(E)-GlcNAc- 645.2543 0.77 1288.5TalNAc*-E M. thermoautotrophicus DSM GlcNAc-TalNAc*-EA 726.3032 -1.10 725.31053 M. stadmanae DSM 3091 Not digested - - -TalA is responsible for cell septum cleavage to complete cell division

[0193] The widespread distribution and conservation of TalA in walled archaea indicates a role inan important cellular process requiring arcPG cleavage, such as cell separation at the end of cytokinesis (1-3). To investigate the function of TalA, antibodies were raised against the C-terminal fragment of M.smithii TalA (Figure 15) and studied its subcellular localization pattern during the M. smithii cell cycle byimmunolabelling (Figure 4). A quantitative analysis was performed with several hundreds of immunolabeledM. smithii cells imaged by conventional epifluorescence microscopy. Cells were divided into three classesfor analysis: (i) non-constricting, (ii) slightly constricting, and (iii) those who had nearly completed division.It was consistently observed strong fluorescent signal at cell septa. In slightly constricting cells, as well ascells that almost completed constriction, fluorescent signal was observed both at the septation plane and at the future division sites in the perspective daughter cells. (Figure 4A). Analysis of more than 1000 cells and mapping of the average fluorescence confirmed the observation that TalA is present at the septum and moves laterally to the new division sites (Figure 4B). To gain further insight into the intracellular localization of TalA, the cells were imaged by 3-dimensional (3D) super-resolution microscopy (Figure 4C). In the non-constricting M. smithii cells, TalA forms a discontinuous ring-like structure at the septation plane (Figure4C, left panel). At later division stages, a smaller TalA ring corresponds with the current septation plane, and two new rings appear in the perspective daughter cells, at the site of the new septation planes (Figure 4C, middle and right panel). Interestingly, a similar localization pattern has been observed in Streptococcus pneumoniae, an ovococcoid bacterium where the hydrolase PcsB localizes to equators and division septa (19)

[0194] To further support the function of TalA as septum hydrolase, a ΔtalA mutant wasconstructed in Methanothermobacter thermoautotrophicus, a close relative of M. smithii and the onlyspecies of walled archaea for which genetic manipulation was recently made available (20, 21). In phasecontrast microscopy, the wild type (WT) naturally forms short chains (Figure 5A). However, the ΔtalA mutantformed much longer filaments compared to WT (Figure 5A), comparable to phenotypes observed inhydrolase mutants of the rod-shaped bacterium B. subtilis (18). To quantify the increase in filamentation,the length of hundreds of filaments was measured for both WT and ΔtalA mutant using the Fiji-4 SNT plugin(Figure 5B). The three WT replicates had a small distribution of filament lengths ranging from 3 to 17µmand means ranging from 9.2 to 10.0µm. In contrast, the ΔtalA mutant displayed highly variable filamentlengths ranging from 3 to 50µm and means ranging from 18 to 28µm (Figure 5B). Mann-Whittney tests were performed between each WT and mutant replicate, and all showed statistical significance with p<e- 16 (Table 2).

[0195] Table 2. Statistical analysis of WT vs ^TalA mutants^TalA1 ^TalA2 ^TalA3 WT1 WT2 WT3^TalA1 - 0.0003476 0.158 < e-16 < e-16 < e-16^TalA2 0.0003476 - 2,11E-04 < e-16 < e-16 < e-16^TalA3 0.158 2,11E-04 - < e-16 < e-16 < e-16WT1 < e-16 < e-16 < e-16 - 0.0001221 0.1556WT2 < e-16 < e-16 < e-16 0.0001221 - 0.1336WT3 < e-16 < e-16 < e-16 0.1556 0.1336 -

[0196] These results show that the absence of TalA leads to failure in cell separation after tocomplete cytokinesis, supporting its function as the main septum hydrolase in walled archaea. Identification and characterization of TalA

[0197] TalA, the first septum hydrolase specific to archaeal PG has been identified andcharacterized. TalA has a previously undescribed Talosaminidase activity, and efficiently cleaves arcPG from diverse walled archaea.

[0198] The discovery of TalA overcomes a major technological lock, enabling all futureinvestigations of arcPG, akin to what was achieved in the past decades following the discovery ofmuramidases in bacteria. TalA revealed a conserved modification on TalNAc that could not be observedby the chemical methods used in the past. These used in fact harsh acidic and basic hydrolysis required toisolate the arcPG disaccharide which destroy N-acetyl groups, followed by artificial re-N-Acetylation of thedisaccharide before GC-MS analysis (7,11). The previously reported N-acetyl group on talosaminuronicacid is instead a previously undescribed chemical modification conserved in arcPG.

[0199] Walled archaeal methanogens are emerging new experimental models in the nascent fieldof archaeal cell biology (22). Together with the recent availability of the first genetic tools for walled archaea, TalA can now be used to identify and characterize the major processes and machineries involved in cell growth and division, which are presently vastly unknown. These include additional hydrolases, equivalents bacterial PBPs, the full pathway of arcPG synthesis, and notably the still elusive enzyme making TalNac*. Their identification will significantly increase knowledge on fundamental biological processes in walled archaea and eventually lead to understanding the puzzling evolutionary origins of arcPG.

[0200] Finally, methane is one of the most potent greenhouse gases, and walled archaealmethanogens represent an important and yet understudied component of diverse anaerobic communities,including the animal and human gastrointestinal tracts. Hence, our findings can drive the development ofmitigation strategies, with broad implications in the ecology and health fields. Identification of candidate arcPG hydrolases

[0201] A collection of the descriptions of 19,632 domains was downloaded from the Pfamdatabase (March 2022). Then, 703 domains were selected with descriptions associated to generic cell wall or hydrolase activity (“cell wall”, “murein”, “peptidoglycan”, "hydrolase", "carboxypeptidase", "amidase", "glucanase", "endopeptidase", "muramidase", "glycosylase", "glucosaminidase"), or specific domains known from the literature to be associated to cell wall hydrolases (“SLH”, “SH3_”, Glyco_hydro","Peptidase_S11", "Peptidase_S13", "VanY", "Peptidase_M20", "Beta-lactamase2", "Peptidase_M15_4", "fn3_3", "Csd3_N", "SLT", "Transgly_assoc", "Transglycosylas", "CarboxypepD_reg","Big_", "Beta_helix"). the proteome of M. smithii was scanned using the entire Pfam collection as HMMprofile database, using hmmscan from HMMER v3.3.2 suite. The 84 proteins that contained at least one of the 703 domains were extracted. Next, the subcellular localization of each protein of the proteome of M.smithii was predicted using Psortb (17). The 51 proteins with annotation “Cell Wall”, “Extracellular” and“Unknown” were kept. Among these, the proteins that were detected in the MS analysis after the zymogram,and ranging between 30 kDa and 180 kDa were selected, leading to a final list of 11 candidates.Growth of bacterial and archaeal strains

[0202] Bacterial and archaeal strains used in this work are listed in Table 3.Table 3. Bacterial strains used in this study.

[0203] E. coli strains were genetically manipulated using standard laboratory procedures (23).When needed, the following compounds were added to E. coli cultures at the following concentrations:chloramphenicol – 30 ug / ml (liquid media) or 25 ug / ml (solid media), diaminopimelic acid – 300 µM,ampicillin 100ug / ml.

[0204] M. smithii was grown as described previously (24). Briefly, an adapted DSMZ 141methanogen medium was used which was made anaerobic by boiling, cooling under nitrogen flux before adding reducing agents and sealing in serum bottles (40ml in 100ml serum bottles). Cultures were inoculated at a 1 / 10 dilution from an actively growing culture and grown with agitation at 180rpm at 37oC for between 3-5 days until turbid.

[0205] M. thermautotrophicus was manipulated as described previously (20, 21, 25), with slightmodifications. For the mineral medium, a different composition of the trace elements solution was used, containing per liter 10 ml of 25% HCl, 1.5 g of FeCl2·•4H2O, 70 mg of ZnCl, 100 mg of MnCl2·•4H2O, 6 mg of H2BO4, 190 mg of CoCl2·•6H2O, 2 mg ofCuCl2·•2H2O, 24 mg of NiCl·2•6H2O and 36 mg of Na2MoO4·•H2O; 10 ml of this solution was added per liter of mineral medium. Na2S was systematically used as a reducer, even in liquid media, sometimes replaced by 0.5 g / l of Na2S2O3. For plate cultures gaseous H2S was replaced by an open tube containing 10 ml of 60 g / l Na2S·•H2O solution. When needed,250 µug / ml of neomycin was added to M. thermautotrophicus cultures. The GP Campus anaerobic chamber(Jacomex) filled with a H2 / CO2 / N2 (5% / 5% / 90%) mixture was used for M. thermautotrophicus manipulation.

[0206] SAB medium (modified): MgSO4·7H2O 0.8 g, KH2PO40.5 g, K2HPO40.5 g, CaCl2·6H2O0.05 g, NaCl 1.5 g, NH4Cl 1 g, Na2S·9H2O 0.3 g, sodium acetate 1 g, yeast extract 2 g, 5mM valeric acid, 5 mM isovaleric acid, 5 mM 2-methylbutyric acid, 6 mM isobutyric acid, 5 mM 2-methyl valeric acid, 1000x TE solution 1 ml in final volume of 1000ml. The SAB-TE solution (TES2) contained the following: nitrilotriacetic acid 1.5 g, NiCl2·6H2O 15 g, KCl 0.5 g, MnSO4·7H2O 6 g, ZnSO4·7H2O 1 g, CuSO41.278 g, KAl(SO4)2·12H2O 2 mg, H3BO370 mg, CoSO4·7H2O 40 mg, Na2MoO3·H2O 5 g, Na2SeO3·5H2O 30mg, Na2WO4·2H2O 40 mg, FeSO4·7H2O 9 g in 1000 ml final volume. After anaerobization and autoclaving the medium, L-Cysteine-HCl·H2O 0.5 g / l, 4 M methanol 20 ml / l, 8 M sodium format 20 ml / l, 10% NaHCO3 20 ml / l, 2% Na2S·9H2O 20 ml / l and vitamin solution 15 ml / l (Wolf´s vitamin solution) must be added to the serum bottles. The pH in the serum bottles was adjusted to 7.5 with 10 M KOH.

[0207] Adapted DSMZ 141 medium: 0.17 g KCl, 2 g MgCl2·6H2O, 0.125 g NH4Cl, 0.053 g CaCl2,0.055 g KH2PO4, 0.84 g MgSO4, 3 g NaCl, 0.5 g Na-acetate, 1 g yeast extract, 5 mL of trace element solution, 1mL of FeII(NH4)2(SO4)2·6H2O solution, 50 μl of Na-resazurin solution (0.7 mg / ml), and was filled up with 480 ml of ddH2O. The medium was brought to boil and was boiled for 5 min. After the temperature descended to 50 °C, 5 ml of vitamin solution, 2.5 g NaHCO3, 0.25g L-Cystein-HCl and 0.1 g Na2S·H2O were added. The pH was adjusted to 7 with HCl and the medium was filled up with ddH2O to a final volume of 500 ml. The medium was flushed with CO2 and transferred with a glass syringe into 100 mLserum bottles. 40 ml of the medium were aliquoted into serum bottles and sealed with blue rubber stoppers(20 mm, Bellco) and open-top aluminum caps (9.5 mm opening, Merck Group).

[0208] M. stadmanae media: 0.5 g / l KH2PO4, 0.4 g / l MgSO4·7H2O, 5 g / l NaCl, 1 g / l NH4Cl, 0.05g / l CaCl2·2H2O, 1.5 g / l Na-acetate, 2 g / l Yeast extract, 2 g / l Tryptone, 2 g / l Peptone, 2 g / l Casamino acids, 1ml / l Selenite-tungstane solution, 1ml / l Na-resazurin solution (0.1 w / v), 10ml / l modified Wolin’s mineral solution (141), 4 g / l NaHCO3, 0.5 g / l Na2S·9H2O, 20ml / l Wolins vitamin solution, 2 g / l TMA, 5ml / l Methanol. All ingredients were dissolved except bicarbonate, vitamins, cysteine, methanol and sulfide and the mediumwas boiled and sparged with gas mixture for 15 – 30 min. Bicarbonate, vitamins, cysteine and sulfide wereadded and the pH adjusted to 7-7.2 before autoclaving. Cultures were inoculated with 10% of a pre-culture,pressurized with a 4:1 H2 / CO2 gas phase and grown for 4 days.

[0209] The sealed serum bottles containing the medium were autoclaved for 20 min at 120 °C.Composition of trace element solution was: 1.5 g Nitrilotriacetic acid, 3g MgSO4·7H2O, 0.585 gMnCl4·4H2O, 1 g NaCl, 0.1 g FeSO4·7H2O, 0.18 g CoSO4·7H2O, 0.1 g CaCl2·2H2O, 0.18 gZnSO4·7H2O, 0.006 g CuSO4, 0.02 g KAl(SO4)2·12H2O, 0.01 g H3BO3, 0.01 g Na2MoO4·2H2O, 0.03 g NiCl2·6H2O, 0.3 mg Na2SeO3·5H2O, 0.4 mg Na2WO4·2H2O in final volume of 1000 ml and pH was adjusted to 7.0 with KOH. Composition of Vitamin solution was: 2mg Biotin, 2mg Folic acid, 10 mg Pyridoxine-HCl, 5 mg Thiamine-HCl, 5mg Riboflavin, 5mg Nicotinic acid, 5mg D-Ca-pantothenate, 0.1 mg Vitamin B12, 5 mg p-Aminobenzoic acid, 5mg Lipoic acid and filled up with ddH2O to a final volume of 1000 ml.

[0210] Methanothermobacter marburgensis medium (MM): NH4Cl 2.1 g, KH2PO 46.8 g, Na2CO33.6 g, 100x TE 10 ml in 1000ml of ddH2O. After anaerobization and autoclaving the medium, 0.5 M Na2S·9H2O 2 ml / l must be added.100x TE solution (TES3) contained: Titriplex I 9 g, add 800 ml ddH2Oand adjust the pH to 6.5 with 5M NaOH solution, then add: MgCl2·6H2O 4 g, FeCl2·4H2O 1 g, CoCl2·6H2O20 mg, NiCl2·6H2O 120 mg, NaMoO4·2H2O 20 mg to a final volume of 1000 ml and put to pH= 7.0 with 1 M NaOH. Culture media

[0211] Considering the nutritional requirements of the different strains, several media were used.SAB medium (26) was used for Methanobacterium bryantii DSM 863 and Methanobrevibacter arboriphilicusDSM 7057, Methanobrevibacter smithii DSM 861 was grown in DSMZ medium 141 as in (24), andMethanothermobacter marburgensis DSM 2133 and Methanobacterium formicicum DSM 1535 were grownin Methanothermobacter marburgensis medium (MM) (27). Methanosphaera stadmanae MCB-3 DSM3091 M. stadmanae media.Peptidoglycan extraction

[0212] Archaeal cells were pelleted by centrifugation at 4000g for 20 minutes. Cell pellets wereresuspended in 10ml of PBS and added dropwise to 10ml of 8% w / v SDS preheated in a boiling water bath and incubated at 100oC for one hour. The cell lysate was cooled to room temperature and cell sacculi were pelleted by ultracentrifugation at 150,000g for two hours. The sacculi were resuspended in 8ml of MiliQH2O and washed a minimum of 8 times by centrifugation at 17,000g to remove residual SDS. The pelletswere then incubated at 37oC in 8ml 50mM Tris-HCl pH7.0 with 100ug / ml alpha-amylase for two hours with constant agitation. Final concentrations of 20mM MgSO4, 10ug / ml Dnase, 50ug / ml Rnase were added, and the digestion was incubated at 37oC for two hours. Final concentrations of 10mM CaCl2 and 100ug / ml trypsin were added, and the digestion was incubated overnight at 37oC with constant agitation. SDS was added at a final concentration of 1% to inactivate the trypsin at 95oC for 15 minutes. After cooling to room temperature, the sacculi were again washed in miliQ H2O four times to remove the SDS. The pellet was resuspended in 8M LiCl and incubated at 37oC with constant agitation for 30 minutes. Sacculi were pelleted by centrifugation at 17,000g for 10 minutes and resuspended in 100mM EDTA to chelate and remove remaining LiCl along with other ions and incubated at 37oC with constant agitation for 15 minutes. Sacculi were pelleted by centrifugation at 17,000g for 10 minutes and washed twice in 8ml MiliQ H2O. Finally, 1ml of 100% acetone was added to the pellet and resuspended by sonication. The pellet was washed twice and lyophilized by freeze drying. Zymogram analysis

[0213] Proteins or cell lysates were separated on 10% Tris-Glycine SDS PAGE gelssupplemented by the addition of 0.5mg / ml purified peptidoglycan. Following electrophoresis, the gel was washed three times by incubation in miliQ H2O for one hour with gentle agitation to remove SDS from the gel. Proteins were then renatured within the gel by incubation in refolding buffer (50mM Tris-HCl, pH750mM NaCl, 0.2% Triton X-100) at 37oC overnight. The gel was then washed 2 times in MiliQ H2O and stained for one hour with 0.1% w / v Methylene blue in 0.01% w / v KOH and de-stained with MiliQ H2O. Proteomic LC-MS / MS analysis

[0214] Bands of hydrolytic activity were cut from the gel and cut and washed several times in 50mM ammonium bicarbonate (ABC), acetonitrile (1:1) for 15 min at 37 °C. Disulfide bonds were reducedwith 10mM DTT (43815 - Sigma, St Louis, Missouri, USA) and cysteine alkylated with 55mMchloroacetamide. Trypsin (V5111 - Promega, Madison, Wisconsin, USA) digestion was performedovernight at 37 °C in 50 mM ABC. Peptides were extracted from the gel by two incubations in 50mM ABC,ACN, formic acid (FA) (50 : 50 : 0.5) for 15 min at RT. After ACN evaporation in a Speed-Vac, resultingpeptides were desalted with stage-tip (Rappsilber et al., 2007) using C18 Empore disc and eluted with 80% ACN, 0.1 % FA. Peptides were resuspended in 2% acetonitrile, 0.1% formic acid prior to LC-MS / MS analysis.

[0215] A nanochromatographic system (Proxeon EASY-nLC 1200- Thermo Fisher Scientific,Waltham, Massachusetts, USA) was coupled on-line to a Q ExactiveTM Plus Mass Spectrometer (ThermoFisher Scientific) using an integrated column oven (PRSO-V1 - Sonation GmbH, Biberach, germany). Foreach sample, peptides were loaded into a capillary column picotip silica emitter tip (home-made column, 40cm x 75 µm ID, 1.9 μm particles, 100 Å pore size, Reprosil-Pur Basic C18-HD resin, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) after an equilibration step in 100 % buffer A (H2O, 0.1 % FA).

[0216] Peptides were eluted with a multi-step gradient from 2 to 7 % buffer B (80 % ACN, 0.1 %FA) in 3 min, 7 to 23 % in 52 min, 23 to 45 % in 22 min and 45 to 95 % in 5 min at a flow rate of 250 nL / min over 104 min. Column temperature was set to 60°C.

[0217] MS data were acquired using Xcalibur software using a data-dependent Top 10 methodwith a survey scans (300-1700 m / z) at a resolution of 70,000 and a MS / MS scan (fixed first mass 100 m / z)at a resolution of 17,500. The AGC target and maximum injection time for the survey scans and the MS / MSscans were set to 3E6, 20 ms and 1E6, 60 ms respectively. The isolation window was set to 1.6 m / z andnormalized collision energy fixed to 28 for HCD fragmentation. A minimum AGC target of 1E4 was used foran intensity threshold of 1.7E5. Unassigned precursor ion charge states as well as 1, 7, 8 and >8 charged states were rejected and peptide match was disable. Exclude isotopes was enabled and selected ions were dynamically excluded for 20 seconds.

[0218] Raw data were analyzed using MaxQuant software version 1.6.6.0 (29) using theAndromeda search engine (30). The MS / MS spectra were searched against a UniProt Methanobrevibacter smithii database (download in 14 / 09 / 2021, 1,783 entries). Usual known mass spectrometry contaminants and reversed sequences of all entries were included.

[0219] Andromeda searches were performed choosing trypsin as specific enzyme with amaximum number of two missed cleavages. Possible modifications included carbamidomethylation (Cys, fixed), oxidation (Met, variable), Nter acetylation (variable). The mass tolerance in MS was set to 20 ppm for the first search then 4.5 ppm for the main search and 20 ppm for the MS / MS. Maximum peptide charge was set to seven and seven amino acids were required as minimum peptide length. One unique peptide to the protein group was required for the protein identification. A false discovery rate (FDR) cutoff of 1 % was applied at the peptide and protein levels. Cryo-Electron Microscopy (CryoEM)

[0220] 2 mL of a fresh culture of M. smithii were pelleted, washed in ddH20, re-pelleted, re-suspended in 50 μL of ddH20 and finally diluted 1:10 in ddH20. A solution of bovine serum albumin (BSA)– gold tracer (Aurion) containing 10 nm -diameter colloidal gold particles was added to the suspension with a final ratio of 1:1. Purified M. smithii sacculi with a concentration of 5 mg / mL were pelleted andresuspended in 10 µL of ddH20 with a sonication probe to avoid aggregation and further diluted in ddH20 (1:5). A solution of BSA-gold tracer (Aurion) was added to the suspension with a final ration of 20:1. Samplefreezing: A small amount of the sample was applied to the front (3 µl) and the back (1.2 µl) of carbon-coatedcopper grids (Cu 200 mesh Quantifoil R2 / 2 or Lacey, Oxford Scientific), previously glow discharged 2 mA and 1.5–1.8 × 10−1 mbar for 1 min in a glow discharge system (ELMO, Corduan). The sample was then vitrified in a Leica EMGP system. Briefly, the excess liquid was removed by blotting the grid backsides with filter paper (7-8 s, 30 s pre-blot time, 18 °C, 98% humidity), and then the sample was rapidly frozen in liquid ethane. The grids were stored in liquid nitrogen until image acquisition in the transmission electron microscope. CryoEM imaging: Images were acquired in a Tecnai 20 equipped with a field emission gun and operated at 200 kV (Thermo Fisher Scientific) using a Gatan 626 side entry cryo-holder. They were recorded using the SerialEM software (version 3.7 beta, U. Colorado Boulder, USA) on a Falcon II (FEI, Thermo Fisher Scientific) direct electron detector, with a 14 µm pixel size at nominal magnification of ×29,000, corresponding to a pixel size of 0.349 nm. For high-magnification images, the defocus was -8 μm. DNA manipulations

[0221] All plasmids and primers used in this study are listed in Table 3, 4 and 5 respectively.Cloning was performed using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs).Chemocompetent homemade E. coli DH5α cells (31) were used for transformation of cloning products orplasmids. M. thermautotrophicus genomic DNA was extracted according to a protocol previously describedfor Streptomyces gDNA extraction (32) from stationary phase cultures in mineral medium. Genomic DNAfrom M. smithii was obtained by resuspending a pellet of densely grown culture in ddH2O and boiling it for5 min at 99 °C. Subsequently,1 μL of archaeal suspension was used as template in each 50 μL PCR reaction. PCR reactions for cloning applications were carried out using Phusion HiFi Master Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol. PCR reactions for the control of constructions were carried out using the DreamTaq Green MasterMix (Thermo Fisher Scientific). All primers were provided by Merck. PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey- Nagel). Restriction enzymes were of the FastDigest family of products (Thermo Fisher Scientific) except for FseI (New England Biolabs). Digestion products were isolated on agarose gels and purified with the GeneJet Gel Extraction Kit (Thermo Fisher Scientific). Plasmid isolation was performed with NucleoSpin Plasmid kit (Macherey-Nagel). Sequence in silico manipulation was carried out using SnapGene (GSL Biotech, www.snapgene.com). Primers were designed either with NEBuilder (New England Biolabs, nebuilder.neb.com) or Primer3Plus (33) or manually. All Sanger sequencing was performed by Eurofins. Table 4. Plasmids used in this study.Table 5. Primers used in this study (SEQ ID NO: 12 to 56, respectively).The part of the primer that does not hybridize with the template DNA is indicated by underscoreConstruction of E. coli expression vectors

[0222] Expression vectors for expression of recombinant archaeal proteins in E. coli wereconstructed by linearizing pET22b with restriction enzymes NdeI and XhoI. Primers were designed to include a start codon and remove a stop for each construct detailed in the primer table. Overhangs were included on each primer with homology to the plasmid sequence to facilitate Gibson assembly. The coding sequence for each construct was cloned in frame with a 6xHis tag and stop codon already present on thevector at the C-terminus. Plasmids were confirmed by Sanger sequencing with T7 forward and reverse primers. Alanine replacement of catalytic residues

[0223] Catalytic residues were identified using the TalA protein sequence alignment andsearching for conserved cysteine, histidine and aspartate residues which are known to be the catalytic triad of C71 peptidases (34, 35). Three residues were identified with high levels of conservation across all TalA homologues. Inverse PCR was performed on pET22b_candidate_8_cter using primers mismatching a single codon to the codon with an alanine codon. After inverse PCR, the template plasmid DNA was removed by digestion with DpnI for one hour and the plasmid was re-circularised using Neb Hifi assembly mastermix. The identity of the mutation was confirmed with Sanger sequencing using the T7 reverse primer. Expression of candidate hydrolases and Zymogram analysis

[0224] E. coli BL21(DE3) cells containing plasmids expressing C-terminally 6xHis taggedcandidate hydrolases were cultured overnight at 37oC in LB supplemented with 100ug / ml Ampicillin. Cultures were diluted in 10ml of fresh LB media and grown at 37oC until OD6000.7 and protein expression was induced with 1mM IPTG. Cells were grown for a further four hours before being pelleted at 4000g for 20 minutes. Cells were resuspended in 250μl of PBS pH7.0, 0.1mm beads were added and cells werelysed using a MP FastPrep-24TM 5 G homogenizer (three rounds of 20s with 60s pauses at 4m / s). Thelysate was removed without clarifying and protein LDS sample buffer was added and 10μl was loaded on a zymogram. Purification of recombinant proteins

[0225] E. coli BL21 cells containing plasmids expressing C-terminally 6xHis tagged recombinantTalA derivatives were cultured overnight at 37oC in LB supplemented with 100ug / ml ampicillin. Overnightculture was diluted 1 / 1000 in autoinduction medium as described previously (24) and cultured at 37oC,shaking at 180rpm until OD6000.7. Cultures were transferred to 30oC and grown overnight. Cells were centrifuged at 6000g for 20 minutes, resuspended in buffer A (50mM Tris-HCl pH7.5, 300mM NaCl, 15mM Imidazole, 5% v / v Glycerol) supplemented with protease inhibitors (Roche cOmplete EDTA free) andbenzonase. Cell lysis was achieved using a cell disruptor (ref) at 1.5kbar pressure. Cell debris was removedby high-speed centrifugation at 50,000g for one hour at 4oC. The cell lysate was loaded onto a Co-NTA affinity column (HiTrap talon 5ml GE healthcare) at 10oC. The protein was then eluted using a linear gradient of buffer B (50mM Tris-HCl pH7.5, 300mM NaCl, 500mM imidazole, 5% glycerol). Proteins were further purified by size exclusion chromatography using a Superdex 20016 / 60 using SEC buffer (20mM Tris-HCl pH7.5, 300mM NaCl). Proteins were stored at 4oC, and enzymatic tests were performed on protein less than one week old. Digestion of arcPG with TalA and rp-HPLC

[0226] 250μg of purified sacculi were incubated with 100μg of purified TalACter in 150μl of SECbuffer overnight at 37oC with constant agitation. Samples were centrifuged at 17,000g for 20 minutes toremove undigested sacculi and 150μl of borate buffer (62.5mM H3BO4, 35.9mM Na2[B4O5(OH)4].8H2O) pH9.0. Excess solid sodium borohydride was added, and samples were reduced for 20 minutes at room temperature. The reaction was neutralized by the stepwise addition of phosphoric acid (80% v / v) until thepH reached 3.0. Samples were then used for either rp-HPLC or LC-MS. 200μl was recovered from the PGdigestion and reduction reactions and 190μl was separated by rp-HPLC on a Hypersil gold AQ C18 column (3um particles, 250mm by 4.6mm; Thermofisher) equilibrated in H2O, 0.05% v / v TFA. Samples were eluted from the column with a linear gradient of acetonitrile (ACN) + 0.05% v / v TFA up to 25% ACN over 135 minutes at a flow rate of 0.5ml / minute at 52oC. Tightly associated molecules were eluted with a 10-minute wash at 25% ACN before the column was re-equilibrated with H2O 0.05% TFA for 30 minutes. Eluted molecules were detected using absorbance of UV light at 206nm. LC / MS analysis of talopeptides

[0227] LC-HRMS analyses were performed using an Ultimate 3000 Ultra High-PerformanceChromatography system (UHPLC; Dionex / Thermo Fisher Scientific) coupled to a Q Exactive Orbitrap mass spectrometer (Q Exactive Focus; Thermo Fisher Scientific). The reduced products of digestion by TalA were separated on a Hypersil GOLD aQ C18 analytical column (150 x 2.1 mm, 1.9 µm) with mobile phase A (0.1% FA) and mobile phase B (Acetonitrile containing 0.1% FA). After injection of 10 µL of sample, the elution consisted to a nonlinear gradient from 0 to 12.5% of phase B for 25 min, followed by an increase to 20% B for 5 min with a mobile phase flow rate of 200 µL / min. The column was flushed for 10 min with80% of phase B at 32 min before letting the system equilibrate for 8 min with 100% of phase A. Columntemperature was set to 50°C.

[0228] MS analyses were performed in the positive ion mode, and sequential MS2 experimentswere carried out using data-dependent acquisition method. A normalized collision energy (NCE) of 25% was used for the fragmentation of talopeptides.XCalibur 4.0 software from Thermo Fisher Scientific was used to control the instrument and for data processing. Sequence analysis and phylogeny

[0229] A local databank of Methanomada was assembled containing 697 genomes (626Methanobacteriales, 62 Methanococcales and 9 Methanopyrales). To build a reference phylogeny, exhaustive HMM-based homology searches with the option --cut_ga were carried out using HMM profiles of RNA_pol_Rpb1, RNA_pol_Rpb2 and the Initiation Factor IF-2 from the Pfam database on the Methanomada databank using HMMSEARCH from the HMMER-3.1b2 package. For each protein, only the taxa with at least two markers were kept and homologous sequences were aligned using MAFFT v7.481 with the linsi option and trimmed using BMGE-1.1 with the BLOSUM30 substitution matrix. The resulting trimmed alignments were concatenated into a supermatrix (642 taxa x 2790 amino acid positions). A maximum likelihood tree was generated using IQ-TREE 1.6.12 under the LG+F+R8 model selected according to the BIC criteria, with 1000 ultrafast bootstrap replicates.

[0230] For TalA homology searches, a customized HMM profile was built. For this, the M. smithiiTalA protein was used to search for homologous in a database containing 4016 archaea and 401 bacteriagenomes using BLAST. Given that the peptidase domain is very largely distributed, all the hits containingthe Pfam glycosyl hydrolase domain (DUF4015) were selected, which were aligned and used to build aHidden Markov Model (HMM) profile using HMMBUILD from the HMMER-3.1b2 suite. The HMM model was then used to search both the Methanomada and the Archaea / Bacteria databank. Homologous proteinswere aligned using MAFFT with the auto option, trimmed using trimAl to remove all columns with more than30% of gaps. The maximum likelihood tree of glycosyl hydrolase-containing homologs from Methanomadawas built using IQ-TREE 2.0.7 under the LG+F+R8 model selected according to the BIC criteria, with 1000ultrafast bootstrap replicates. The maximum likelihood tree of glycosyl hydrolase-containing homologs fromArchaea and Bacteria was built using IQ-TREE 2.0.7 under the LG+F+R6 model selected according to theBIC criteria, with 1000 ultrafast bootstrap replicates.

[0231] Finally, domain organization of the glycosyl hydrolase-containing homologs was assessedby scanning all protein sequences for Pfam domains using HMMSCAN and the Pfam database, and then mapped on the different phylogenies using IToL.Antibody production and Immunostaining

[0232] Antibodies were raised against purified TalACter in guinea pigs (Covalab). 50ml of M. smithiiculture OD6000.4 was centrifuged and resuspended in 250μl of PBS and 100μl of 0.1mm glass beads were added. Cells were then lysed using a MP FastPrep-24TM5 G homogenizer (three rounds of 20s with 60spauses at 4m / s). LDS sample loading dye was added and 20μl of cell lysate were separated on a 4-12%Bis-Tris gel. The gel was blotted onto a 0.2um nitrocellulose membrane using an Invitrogen power blotterwith 7 minutes of blotting. Membranes were rinced twice in PBS. Membranes were blocked with 5% w / vskimmed milk in PBS + 0.05% Tween 20 (PBST). Guinea pig anti-TalACter antibody was added at 1 / 1000 in 5% PBST and the blot was stained overnight at 4oC with gentle agitation. After washing with PBST x-animal anti guinea pig IgG linked to horseradish peroxidase was added at 1 / 5000 and stained for one hour at room temperature. After three washes with PBST and a final wash with PBS the blot was revealed with HRP substrate and imaged on a Biorad ChemiDoc MP with chemiluminescence.Cells of M. smithii grown to early exponential phase were harvested by 5 min of centrifugation at 3.5 × g (all centrifugation steps were performed at 3.5 × g). Pellets were washed in PBS buffer, pelleted, fix with 80% ice cold methanol andstored at −20 °C. Cells were rehydrated and washed PBS buffer.

[0233] Cells were pelleted and washed three times in PBS-T (PBS buffer with Tween 20 at 0.1 %(v / v)). Blocking was carried out for 1 h in PBS-T containing 2 % (w / v) bovine serum albumin (blocking solution) at RT. Cells were incubated with a 1:200 dilution of guinea pig polyclonal anti-TalACter antibody for 1 h at RT in blocking solution. Upon incubation with primary antibody samples were pelleted and washed three times in PBS-T, and incubated with a 1:500 dilution of secondary Alexa488-conjugated anti-guinea pig antibody (Invitrogen) in PBS-T for 1 h at RT. Unbound secondary antibody was removed by three washing steps in PBS-T. Finally, cells were resuspended in few μl of PBS and slides were prepared either for super resolution microscopy or epifluorescence microscopy (see below). Three-dimensional structured illumination microscopy (3D SIM) imaging and analysis

[0234] Archaeal cell suspensions were applied on high precision coverslips (No.1.5H, Sigma-Aldrich) coated with 0.01 % (w / v) of Poly-L-Lysin. After letting the cells attach onto the surface of the coverslip for 10 min, residual liquid was removed, 8 μl of antifade mounting medium (Vectashield) were applied and the coverslip was sealed to a slide. SIM was performed on a Zeiss Elyra 7 Lattice SIM microscope (Carl Zeiss, Germany) using Plan-Apochromat 63x / 1.4 Oil DIC M27 (WD 0.19 mm) objectivewith a 1.518 refractive index oil (Carl Zeiss, Germany). The samples were excited with laser at 488 nm andthe emission was detected through emission filter LBF 405 / 488 / 561 / 642. The fluorescence signal wasdetected on a pco.edge 4.2 CL HS sCMOS camera. Raw images are composed of fifteen images per planeper channel (five phases, three angles), and acquired with a Z-distance of 0.11 μm. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed with ZEN Black 3.0 SR software (Carl Zeiss, Germany) using SIM2.

[0235] For further image analysis of SIM image z stacks, Fiji (ImageJ) Version 2.0.0-rc-68 / 1.52iwas used. Namely, a color was assigned to the fluorescent channel, stacks were fused to a single image (z projection, maximum intensity), movies were created via 3D projection and images at different rotationangles were extracted. Regions of interest were cut out and, for uniformity, placed on a black rectangularbackground. Figures were compiled using Adobe Illustrator 2022 (Adobe Systems Inc. USA). Morphometric and fluorescence measurements

[0236] 2 μl of immunolabelled M.smithii cells solution together with 1 μl of Vectashield (VectorLabs) were applied to an 1 % (w / v) agarose covered microscopy slide and imaged using a Nikon EclipseNI-U microscope equipped with a MFCool camera (Jenoptik). Images were acquired using the ProgRes Capture Pro 2.8.8 software (Jenoptik) and processed using Fiji (ImageJ) Version 2.0.0-rc-68 / 1.52i incombination with plugin MicrobeJ Version 5.13 o (19) - beta. The cell outlines were traced and cell length,width, fluorescence intensity along the cell length and cell shape were measured automatically. Automatic cell recognition was manually double-checked. For the average fluorescence intensity plots cells were automatically grouped into three size classes according to the cell length. Average fluorescence intensity plots were created in MicrobeJ, representative cells were chosen from bigger fields of few and their brightness and contrast were adapted in Fiji. Figures were compiled using and Illustrator 2022 (Adobe Systems Inc. USA).Construction of the talA mutant

[0237] A suicide vector was constructed by cloning the upstream and downstream fragments ofthe talA gene (amplified with, respectively, JW361 / JW362 and JW365 / 366 primer pairs using M. thermautotrophicus gDNA as a template) with the aph resistance marker (amplified from pMVS-V1 plasmid with JW363 / JW364 primer pair) into pMVS-V1 digested with FseI and SfaAI restriction enzymes (the 2555 bp long fragment containing E. coli replicon was used; the double digestion was performed in the Fast Digest Green Buffer), yielding pJW101 plasmid. The vector’s integrity was verified by Sanger sequencing of the insert. This vector was then used to delete the talA gene following the protocol described by Fink and his collaborators (20) with minor modifications (use of electrocompetent (36) MFDpir (37) E. coli conjugativestrain instead of ET12567 and the modifications described in the previous section). Multiple resulting clones were tested by PCR verifying the existence of the junctions between the genome and the resistance marker (JW367 / JW359 primer pair for downstream junction and JW360 / 368 for upstream junction) and the absence of the talA gene (primer pair JW419 / JW420) and a single correct clone was selected for further study. Microscopy and chain length measurement of M. thermoautotrophicum

[0238] A single actively growing culture of both WT and ΔtalA was used to inoculate threeindependent cultures and grown at 60oC overnight with agitation at 180 rpm in fink medium. 100 μl of culturewas removed from each and Hoechst 33342 was added at 1 / 2000 and incubated for 15 minutes at room temperature in the dark.5μl of stained cell suspension was applied to a microscope slide and imaged using a Ziess axioplan 2 microscope equipped with an Axiocam 503 mono camera (Carl Zeiss, Germany). Epifluorescence and phase contrast images were acquired using ZEN lite software (Carl Zeiss, Germany) and processed using Fiji (ImageJ) version: 2.14.0 / 1.54f with SNT plugin installed (https: / / imagej.net / plugins / snt / ). Between 25 and 100 fields of view were acquired in phase contrast for each replicate giving between 111 and 344 measurable cell chains per replicate totaling 713 chains for WT and 512 chains for ΔtalA. Software was not able to detect cell chains accurately and automatically for both WT and mutant but more noticeably in the mutant as long chains did not fall flat in the focal plane and therefore can modulate between phase bright and phase dark. To overcome this cell chains were drawn on using a semi-automatic method used to measure neurons (SNT plugin). Several points along the length of a chain were selected and the path between was drawn by the software. Overlapping chains of two were measured when the two chains could be clearly differentiated. Overlapping chains of 3 or more were discarded as it was difficult to differentiate. Path lengths from each image were measured and exported as csv files which were further analyzed using excel and R pairwise Mann-Whittney tests were used tocompare replicates Representative fields of view were acquired for WT and mutant with both phase contrastand Hoechst 33342 to use as representations.

[0239] TalA effect on PG bearing archaea and gut bacteria Microbe Medium TemperatureMethanobrevibacter smithii M141 37Escherischia coli LB 37Bacillus subtilis LB 37Table 6: microbes used in this study and culture conditions I. Cultures- Inoculate serum bottles with all microbes in appropriate mediumCulture Replicates Total volumeMethanobrevibacter smithii 4 80mLEscherischia coli 1 20mLBacillus subtilis 1 20mLTable 7: number of serum bottles per microbial culture (anaerobic) -Incubate cultures at 37°C until stationary phase is reached- Record optical density (OD)II. Resuspension and TalA test^ Inside the anaerobic chamberIf OD < 1 -Dilute to OD=0.2 if necessary- Retrieve 10mL of each microbial culture and centrifugate 5000g 10min- Discard supernatant- Resuspend the pellet in 1mL of anaerobic sterile PBS- Transfer resuspension in a sterile Hungate tube- Add 1mL of PBS in the tube (total volume=2mL, OD=1)- Repeat x times per microbial culture with OD < 1If OD > 1 -Dilute to OD = 1- Centrifugate 2mL 5000g 10min- Discard supernatant- Resuspend the pellet in 1mL of anaerobic sterile PBS- Transfer resuspension in a sterile Hungate tube- Add 1mL of PBS in the tube (total volume=2mL, OD=1)- Repeat x times per microbial culture with OD > 1- Add 12.5uL TalA (0.05mg) in resuspended cultures (0.025mg / mL)- Add 25uL TalA (0.1mg) in resuspended cultures (0.05mg / mL)- In remaining tubes, add 12.5 or 25uL of TalA buffer- Add 1.25 or 2.5uL of a 10mM DTT solution to reach 1mM for 15.5 or 25uL (as well as thecontrols) III. Monitoring- Incubate the tubes at 37°C with gentle agitation (80rpm)- Record optical density every 30min- When no OD change is recorded, keep cells for live / dead staining

[0240] Results are shown in Tables 8, 9, 10-12 and Figures 16-18. ArmA stands for TalA as anequivalent synonym in the Tables below and throughout the present description, figures and application.

[0241] Table 8 (below): Raw optical density measured at 600nm over timetime (hour) 0 0,5 1 1,5 2 3 4M-smithii 0,77 0,75 0,75 0,75 0,75 0,75 0,75M-smithii + buffer 0,71 0,71 0,71 0,71 0,71 0,7 0,69M-smithii + 100ug ArmA 0,77 0,4 0,3 0,27 0,27 0,21 0,18M-smithii + 50ug ArmA 0,92 0,6 0,5 0,47 0,47 0,4 0,37E-coli 1,12 1,09 1,08 1,05 1,04 1 1,02E-coli + buffer 1,18 1,13 1,11 1,06 1,02 0,95 1,03E-coli + 100ug ArmA 1,13 1,06 1,05 1,04 1,06 0,98 0,97E-coli + 50ug ArmA 1,11 1,09 1,1 1,05 1 0,93 0,98B-subtilis 1,1 1,08 1,04 0,99 1 0,9 0,9B-subtilis + buffer 1,1 1,09 1 0,93 0,97 0,89 0,9B-subtilis + 100ug ArmA 1,2 1,2 1,14 1,07 1,06 0,97 0,96B-subtilis + 50ug ArmA 1,12 1,09 1,07 0,99 0,92 0,91 0,93

[0242] Table 9 (below): Percentage of initial optical density over timeM-smithii 100 97,4025974 97,4025974 97,4025974 97,4025974 97,4025974 97,4025974M-smithii + buffer 100 100 100 100 100 98,5915493 97,1830986M-smithii + 100ug ArmA 100 51,9480519 38,961039 35,0649351 35,0649351 27,2727273 23,3766234M-smithii + 50ug ArmA 100 65,2173913 54,3478261 51,0869565 51,0869565 43,4782609 40,2173913E-coli 100 97,3214286 96,4285714 93,75 92,8571429 89,2857143 91,0714286E-coli + buffer 100 95,7627119 94,0677966 89,8305085 86,440678 80,5084746 87,2881356E-coli + 100ug ArmA 100 93,8053097 92,920354 92,0353982 93,8053097 86,7256637 85,840708E-coli + 50ug ArmA 100 98,1981982 99,0990991 94,5945946 90,0900901 83,7837838 88,2882883B-subtilis 100 98,1818182 94,5454545 90 90,9090909 81,8181818 81,8181818B-subtilis + buffer 100 99,0909091 90,9090909 84,5454545 88,1818182 80,9090909 81,8181818B-subtilis + 100ug ArmA 100 100 95 89,1666667 88,3333333 80,8333333 80B-subtilis + 50ug ArmA 100 97,3214286 95,5357143 88,3928571 82,1428571 81,25 83,0357143

[0243] Table 10 (below): Percentage of initial optical density over time for M. smithii PS –plotted on Figure 16 time ctrl buffer 100ug ArmA 50ug ArmA0 100 100 100 1000,5 97,4025974 100 51,9480519 65,21739131 97,4025974 100 38,961039 54,34782611,5 97,4025974 100 35,0649351 51,08695652 97,4025974 100 35,0649351 51,08695653 97,4025974 98,5915493 27,2727273 43,47826094 97,4025974 97,1830986 23,3766234 40,2173913

[0244] Table 11 (below): Percentage of initial optical density over time for E. coli DH5alpha – plotted on Figure 17time ctrl buffer 100ug ArmA 50ug ArmA0 100 100 100 1000,5 97,3214286 95,7627119 93,8053097 98,19819821 96,4285714 94,0677966 92,920354 99,09909911,5 93,75 89,8305085 92,0353982 94,59459462 92,8571429 86,440678 93,8053097 90,09009013 89,2857143 80,5084746 86,7256637 83,78378384 91,0714286 87,2881356 85,840708 88,2882883

[0245] Table 12 (below): Percentage of initial optical density over time for B. subtilis –plotted on Figure 18 time ctrl buffer 100ug ArmA 50ug ArmA0 100 100 100 1000,5 98,1818182 99,0909091 100 97,32142861 94,5454545 90,9090909 95 95,53571431,5 90 84,5454545 89,1666667 88,39285712 90,9090909 88,1818182 88,3333333 82,14285713 81,8181818 80,9090909 80,8333333 81,254 81,8181818 81,8181818 80 83,0357143References1. Do, T., Page, J. E. & Walker, S. Uncovering the activities, biological roles, and regulation of bacterial cellwall hydrolases and tailoring enzymes. Journal of Biological Chemistry 295, 3347–3361 (2020).2. Vollmer, W., Joris, B., Charlier, P. & Foster, S. Bacterial peptidoglycan (murein) hydrolases. FEMS MicrobiolRev 32, 259–286 (2008).3. Rajguru, V., Chatterjee, S., Garde, S. & Reddy, M. Crosslink cleaving enzymes: the smart autolysins thatremodel the bacterial cell wall. Trends in Microbiology Preprint at https: / / doi.org / 10.1016 / j.tim.2023.11.004 (2023).4. 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Claims

CLAIMS 1. An isolated and purified Talosaminidase protein or polypeptide.

2. The protein or polypeptide of claim 1, wherein the protein or polypeptide:a. has an amino acid sequence having at least 90% identity with any one of SEQ ID NO:1 to 8, and / or b. comprises or consists of the sequence of any one of SEQ ID NO: 1 to 8, and / orc. differs from any sequence of b., by one or several conservative amino acidsubstitution(s), and / or d. has at least 90% identity with SEQ ID NO:1 through SEQ ID NO:8, ore. is a fragment, especially a fragment of contiguous amino-acid residues of at least 20amino-acid residues, of any one of the sequences defined in a. to d.

3. The Talosaminidase protein or polypeptide of any one of claim 1 or claim 2, which is asequence or fragment containing archaeal peptidoglycan (PG) binding and / or glycosyl hydrolase and / or peptidase activity(ies).

4. The Talosaminidase protein or polypeptide of any one of claim 1 to claim 3, whichencompasses one or more functional domains selected among: a glycosyl hydrolase domain, an archaeal peptidoglycan (PG) binding domain such as an arcPG-binding pseudomurin binding repeat (PMBR) domain, a peptidase domain such as a Cysteine proteinase C71 family peptidase domain.

5. The Talosaminidase protein or polypeptide of any one of claim 1 to claim 4, whichencompasses, in this order from its N-terminal extremity to its C-terminal extremity: a glycosyl hydrolase domain, one or more an archaeal peptidoglycan (PG) binding domain(s) such as an arcPG-binding pseudomurin binding repeat (PMBR) domain(s) and a peptidase domain such as a Cysteine proteinase C71 family peptidase domain.

6. The Talosaminidase protein or polypeptide of any one of claim 1 to claim 5, which furtherencompasses one or more IG-like domain(s), preferably at the N-terminal extremity of the protein or polypeptide.

7. The Talosaminidase protein or polypeptide of any one of claim 1 to claim 6, which has adual enzymatic activity encompassing both of glycosyl hydrolase and peptidase activities.

8. The Talosaminidase protein or polypeptide of any one of claim 1 to claim 7, which hasthe property to cleave a glycosidic bond between position 1 of a TalNAc moiety and position 3 of aGlcNAc moiety, in particular a b(1,3) linkage between position 1 of a TalNAc moiety and position 3 of a GlcNAc moiety, especially as part of its glycosyl hydrolase activity.

9. The Talosaminidase protein or polypeptide of any one of claim 1 to claim 8, which hasthe property to digest peptidoglycan (PG) of archeal type, in particular specifically digest peptidoglycan(PG) of archeal type.

10. The Talosaminidase protein or polypeptide of any one of claim 1 to claim 9, wherein theprotein or polypeptide is a methanogen Talosaminidase protein or polypeptide.

11. An in vitro or ex vivo method of cleaving an archaeal peptidoglycan (arcPG) comprisingcontacting the Talosaminidase protein or polypeptide of any one of claim 1 to claim 10 with the arcPG.

12. The in vitro or ex vivo method of claim 11, wherein the archaeal peptidoglycan (arcPG) isa methanogen peptidoglycan.

13. A pharmaceutical or veterinary composition comprising the Talosaminidase protein orpolypeptide of any one of claims 1 to 10.

14. Talosaminidase protein or polypeptide of any one of claims 1 to 10, or pharmaceutical orveterinary composition of claim 13, for bactericidal use.

15. The Talosaminidase protein or polypeptide or pharmaceutical or veterinary composition ofclaim 14, for use according to claim 14, which is for lysing archaea bacteria, notably through lysis of their cell walls.

16. The Talosaminidase protein or polypeptide or pharmaceutical or veterinary composition ofclaim 15, for use according to claim 15, wherein the archaea are methanogens.

17. The Talosaminidase protein or polypeptide or pharmaceutical or veterinary composition ofany one of claims 14 to 16, for use according to any one of claims 14 to 16, in treating a ruminant.

18. The Talosaminidase protein or polypeptide or pharmaceutical or veterinary composition ofany one of claims 14 to 16, for use according to any one of claims 14 to 16, in treating a human.

19. An isolated nucleic acid encoding the Talosaminidase protein or polypeptide of any one ofclaims 1 to 10.

20. An isolated nucleic acid which has at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

9.

21. A vector comprising a nucleic acid encoding the Talosaminidase protein or polypeptide ofany one of claims 1 to 10, or comprising or consisting of a nucleic acid of claim 19 or 20.

22. The vector of claim 21, wherein said vector is an expression vector.

23. The expression vector of claim 22, wherein said expression vector is a bacterial expressionvector.

24. The vector of any one of claims 21 to 23, which comprises or consists of or has thesequence of SEQ ID NO: 10 or 11.

25. A host cell comprising the expression vector of any one of claims 21 to 24.

26. The host cell of claim 25, wherein said host cell is a bacterial cell.

27. An in vitro or ex vivo method for expressing Talosaminidase protein or polypeptidecomprising culturing the host cell of claim 26 under conditions suitable for expression of the Talosaminidase protein or polypeptide encoded by the expression vector.

28. The in vitro or ex vivo method of claim 27, further comprising purifying the Talosaminidaseprotein produced by the host cell.

29. An antibody that binds to the protein or polypeptide of any one of claim 1 to claim 10.

30. A method of reducing bacterial methane production from a subject animal comprisingadministering a pharmaceutical or veterinary composition comprising the Talosaminidase protein or polypeptide of any one of claims 1 to 10 to a subject animal comprising archaea in their digestive tract, wherein the archaea are methanogens.

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