WO2018185222A1

WO2018185222A1 - Aminoacyl trna synthetases and orthogonal trnas

Info

Publication number: WO2018185222A1
Application number: PCT/EP2018/058731
Authority: WO
Inventors: Birgit Wiltschi; Tea PAVKOV KELLER; Patrik Fladischer
Original assignee: Acib Gmbh - Austrian Centre Of Industrial Biotechnology
Priority date: 2017-04-05
Filing date: 2018-04-05
Publication date: 2018-10-11

Abstract

The present invention relates to polynucleotides capable of catalyzing the aminoacylation of its cognate tRNA with aromatic or aliphatic amino acids to form an aminoacyl-tRNA, polypeptides encoded by such polynucleotides, vectors comprising such polynucleotides, host cells comprising such vectors, pairs of aminoacyl tRNA synthetases and cognate tRNAs, use of such compounds for catalyzing the aminoacylation of aromatic or aliphatic amino acids with corresponding tRNA to form an aminoacyl-tRNA, and methods for catalyzing the aminoacylation of cognate tRNA with aromatic or aliphatic amino acids to form an aminoacyl-tRNA.

Description

Aminoacyl tRNA synthetases and orthogonal tRNAs

The present invention relates to polynucleotides encoding polypeptides capable of catalyzing the aminoacylation of its cognate tRNA with aromatic or aliphatic amino acids to form an aminoacyl-tRNA, polypeptides encoded by such polynucleotides, vectors comprising such polynucleotides, host cells comprising such vectors, pairs of aminoacyl tRNA synthetases and cognate tRNAs, use of such compounds for catalyzing the aminoacylation of its cognate tRNA with aromatic or aliphatic amino acids to form an aminoacyl-tRNA, and methods for catalyzing the aminoacylation of its cognate tRNA with aromatic or aliphatic amino acids to form an aminoacyl-tRNA.

With a few exceptions, all known organisms use the same set of 20 canonical amino acids (cAAs) prescribed by the genetic code for protein biosynthesis. As a result, the chemical diversity in proteins is confined to this small and defined set of cAAs (Wan et al., Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics (2014), 1844 (6): 1059-1070). Proteins implement a remarkable range of functions, but often they do not offer the chemistries that are desirable for biotechnological applications. These limitations may be overcome using non-canonical amino acids (ncAAs), which are a very diverse group of compounds. Though naturally not participating in protein translation, ncAAs offer a broad spectrum of side chain chemistries and many of them occur in nature (Walsh et al., Angewandte Chemie Int Ed (2013), 52 (28): 7098-7124). Currently, there are several approaches available to incorporate these ncAAs into proteins to exploit their broad structural and functional repertoire.

Tools for the site-specific incorporation of ncAAs into target proteins ae known in the art (Wang et al., Science (2001 ), 292 (5516): 498-500; Hohsaka et al., J Biol Chem (2010), 6 (6): 809-815). To do so, an engineered aminoacyl-tRNA synthetase (aaRS) is needed that accepts exclusively the ncAA and charges it on its cognate tRNA. For example, a tRNA suppressing a stop codon may be used. This methodology is known as stop codon suppression (SCS) (Young et al., J Biol Chem (2010), 285 (15): 1 1039-1 1044). For SCS it is important that the pair consisting of the ncAA-specific aaRS and the tRNA_CuA is orthogonal (o-pair), which means it does not interfere with the endogenous translation system of the host (Wang (2001 ), loc. cit.). In early years, many of the o-pairs developed were based on the archaeal TyrRS/tRNA_CuA^Tyr from Methanocaldococus janaschii (Mj) (Ryu et al., Nature Methods (2006), 3 (4): 263-265). The Mj o-pairs have been vastly used for the incorporation of a whole set of different ncAAs (Dumas et al., Chemical Science (2015), 6 (1 ): 50-59). A very efficient naturally occurring system for the reassignment of the TAG codon was first discovered in the archeaeal Methanosarcinaceae species. These archaea incorporate the lysine analog pyrrolysine (Pyl) into several methylamine methyltransferases in response to an in-frame TAG codon when facing certain environmental conditions (Srinivasan et al., Science (2002), 296 (5572): 1459-1462). The decoding of Pyl in Methanosarcinaceae is carried out by pyrrolysyl-tRNA synthetase (PylRS) that specifically charges Pyl onto its cognate tRNA_CuA^Pyl- The charged Pyl- tRNA_CuA^Pyl is capable of suppressing an in-frame TAG codon by the incorporation of Pyl at that position (Krzycki et al., Curr Op Microbiol (2005), 8 (6): 706-712). Since the discovery of the Pyl decoding machinery in Methanosarcinaceae species, Pyl containing proteins were also found in the gram-positive bacterium Desulfitobacte um hafniense, in the human intestinal bacterium Bilophila wadsworthia, as well as selected Clostridium and Deltabacte a species (Gaston et al., Curr Op Microbiol (201 1 ), 14 (3): 342-349). Even though several natural Pyl decoding machineries were identified, only a few of them have been established for site-specific modification of proteins applying the SCS approach. Among these are the highly homologous PylRS/tRNA_CuA o-pairs from Methanosarcina mazei (Mm) (Nozawa et al., Nature (2009), 457 (7233): 1 163-1 167) and Methanosarcina barkeri {Mb) (Polycarpo et al., FEBS Letters (2006), 580 (28-29): 6695-6670). An invaluable feature of the PylRSs from Mm and Mb for protein engineering by SCS is the natural substrate promiscuity of both enzymes for a stunning set of Pyl derivatives. To understand the molecular function of the enzyme (Yanagisawa et al., Chem & Biol (2008), 15 (1 1 ): 1 187-1 197), the crystal structure of the catalytic domain of MmPylRS was solved (Kavran et al., PNAS USA (2007), 104 (27): 1 1268-1 1273). The crystal structure facilitated the engineering of PylRSs for new substrates. In this way, the substrate scope of /WmPyIRS and M)PylRS was extended to other Pyl and lysine analogs (Wan (2014), loc. cit.). Furthermore, MmPyIRS was engineered to accept non-canonical aromatic amino acids (Wang et al., Mol BioSystems (201 1 ), 7 (3): 714-717).

Meanwhile, /WmPyIRS and ΛtePylRS are used routinely for the site-specific incorporation of a palette of ncAAs in E. coli. Moreover, the archaeal PylRS/tRNA_CuA pairs are orthogonal not only in E. coli but also in other organisms, such as yeasts (Hancock et al., J Am Chem Soc (2010), 132 (42): 14819-14824) and mammalian cells (Mukai et al., Biochem Biophys Res Comm (2008), 371 (4): 818-822). Besides the o-pairs from Mm and Mb, a proof of concept on the successful exploitation of the bacterial PylRS/tRNA_CuA^Pyl from Desulfitobacterium hafniense for site-specific incorporation of several Pyl analogs into target proteins in E. coli were published (Herring et al., Nucleic Acid Res(2007), 35 (4): 1270-1278; Katayama et al., Bioscience, Biotechnol, Biochem (2012), 76 (1 ): 205-208). However, a broadly applicable orthogonal DftPylRS/DMRNAcu_A pair has not yet been reported.

O-pairs derived from Pyl decoding machineries provide a useful tool for the site-specific modification of proteins by SCS. Even though the Mm and Mb o-pairs are well established, more o-pairs could be recruited for their application in SCS. Alternative or more advantageous aminoacyl tRNA synthetases, alone or as part of o-pairs might broaden the toolbox for SCS and may spark improvements with regard to efficiencies or substrate scopes. Accordingly, the technical problem underlying the present invention was to comply with the needs set out above. The technical problem has been solved by means and methods as described herein as defined in the claims.

The present invention addresses the technical problem by providing polynucleotides comprising a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence according to SEQ ID NO: 1 or 3, wherein said polynucleotide encodes an aminoacyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA,

wherein said nucleotide sequence being at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence according to SEQ ID NO: 1 or 3 is codon-optimized to a selected host cell, and

wherein said host cell is not Candidatus Methanomethylophilus alvus Mx1201 Ca (^'Candidatus Methanomethylophilus alvus Mx1201 Ca" also referred to herein as "Mma"). As has been found in context with the present invention, aminoacyl tRNA synthetases (also referred to herein as "aaRS") as described and provided herein, e.g. those for pyrrolysine (pyrrolysyl tRNA synthetases, also referred to herein as "PylRS") particularly from Candidatus Methanomethylophilus alvus (Mma) exhibit highly efficient capability to introduce aliphatic amino acids, particularly lysine derivatives, into polypeptides during translation. In context with the present invention, it is also envisaged that aminoacyl tRNA synthetases as described and provided herein are further or alternatively capable of introducing aromatic amino acids into polypeptides during translation.

As found in context with the present invention and different to Pyl decoding bacteria or other archaea known in the art, only 1.6% of the genes of Mma terminate in a TAG amber stop codon. In context with the present invention, this new orthogonal MmaPylRS/MmatRNAcuA pair was inter alia and exemplarily expressed in E. coli and identified as a potent target for the site-specific incorporation of Pyl and its analogs. As generally known in the art, aminoacyl tRNA synthetases (aaRS) are capable of attaching amino acids onto tRNAs cognate to the respective aaRS. As known in the art and in context with the present invention, "cognate tRNA" may mean that such tRNA is usually specifically recognized or preferred by the respective corresponding aaRS. This attachment step corresponds to an esterification step which is catalyzed by the aaRS, so that the respective tRNA is charged (or aminoacylated, attached, linked, loaded, etc.) with the respective amino acid to form an aminoacyl-tRNA. Accordingly, as used herein, this attachment step may also be referred to as charging, loading, esterification, aminoacylation, or the like as understood by the person of skill in the art. tRNAs comprise an anticodon which are capable of identifying corresponding codons on the mRNA, usually either specific/base-by-base, or by wobble base pairing as known in the art. Also, as known in the art, usually tRNAs are specific for or prefer a particular amino acid with which the cognate tRNA may be charged by a corresponding aaRS. In one embodiment of the present invention, the amino acids which are charged onto the cognate tRNA are non-canonical.

"Aliphatic" amino acids as used herein comprise canonical aliphatic amino acids such as isoleucine, leucine, methionine, valine, alanine, glycine, and proline (particularly isoleucine, leucine, methionine, and valine), as well as non-canonical amino acids such as ornithine and derivatives, norleucine, methoxinine, S-allyl-L-homocysteine, S- propargyl-L-homocysteine, L-azidohomoalanine, O-allyl-L-homocysteine, O-propargyl-L- homocysteine, S-azidopropyl-L-homocysteine, alanine derivatives carrying saturated and unsaturated C7-C10 side chains, or beta-hydroxy amino acids such as 2-amino-3- (bicyclo[2.2.1]hept-5-en-2-yl)-3-hydroxypropanoic acid.

"Aromatic" amino acids as used herein comprise canonical aromatic amino acids such as histidine, phenylalanine, tryptophan, and tyrosine, as well as non-canonical amino acids such as S-furyl-L-homocysteine, para-azido-L-phenylalanine, para-acetyl-L- phenylalanine, or para-propargyloxy-L-phenylalanine.

In one embodiment of the present invention, the inventive aminoacyl tRNA synthetases as described and provided herein allow aminoacylation of cognate tRNAs having an anticodon matching to a stop codon, preferably amber (UAG), ochre (UAA) or opal (UGA). According to the present invention, for example, the tRNA cognate to Pyl may be charged with Pyl by the enzyme PylRS (which catalyzes aminoacylation of tRNA^Pyl with Pyl) according to the present invention. The tRNA^Pyl has the anticodon CUA (tRNA_CuA), which matches to the amber stop codon UAG. That is, Pyl is encoded by the amber stop codon UAG. In this context, in one embodiment of the present invention, the tRNA corresponding to an aliphatic amino acid which are aminoacylated by the aaRS (e.g., PylRS) encoded by a polynucleotide as described and provided herein may be tRNA^Pyl (tRNAcu_A), and the corresponding aliphatic amino acid may be Pyl or another lysine derivative. In context with the present invention, by expressing an aminoacyl tRNA synthetase (e.g., PylRS) as described and provided herein, preferably together with its orthogonal tRNA (e.g., tRNA^Pyl (tRNA_CuA)) recognizing a stop codon (e.g., amber, ochre or opal; preferably amber) or any sense codon, it is possible to suppress such stop codons as the translation does not stop at the stop codon but the amino acid (e.g., Pyl) corresponding to its cognate tRNA (e.g., tRNA^Pyl (tRNA_CUA)) is introduced into the polypeptide during the translation process. Analogously, for sense codons, it may be possible to introduce a non-canonical amino acid at that site instead of a canonical which is usually encoded by such codon.

Generally, as used herein, the term "catalyzing aminoacylation of (an) amino acid(s) (with its corresponding/cognate tRNA)" means the process of adding an aminoacyl group to a compound by covalently linking (charging, attaching, loading, linking, bonding, or the like) the respective amino acid to the 3' end of the corresponding/cognate tRNA molecule to form an aminoacyl tRNA as known in the art and also shown and exemplified herein. Methods for assessing the capability of a given enzyme (aaRS) to catalyze such aminoacylation steps are known in the art and are described, e.g., in Francklyn et al., Methods (2008), Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases, 44(2), 100-1 18. It is also possible to indirectly or implicitly show successful catalysis of aminoacylation by a given enzyme (aaRS), based on the observation of introduction of a given amino acid (e.g., Pyl) into a polypeptide as this cannot take place without prior aminoacylation of the cognate tRNA with the corresponding amino acid. Also comparisons with negative control samples, e.g., without addition of the tRNA cognate to the respective enzyme (aaRS), allow indirect or implicit conclusion of successful aminoacylation catalysis ability of the enzyme (aaRS) as readily understood by the person skilled in the art. For example, in context with the present invention, a given enzyme (aaRS) may be considered to exhibit aminoacylation ability (i.e. ability to catalyze aminoacylation) if introduction (or incorporation, herein used synonymously in this context) of the corresponding amino acid into a polypeptide can be observed. Introduction or incorporation of an amino acid into a polypeptide may be observed by any method known in the art, comprising inter alia mass spectrometry or Edman degradation as known in the art and also described and exemplified herein. In context with the present invention, this aminoacylation process may preferably be catalyzed by polypeptides acting as aminoacyl tRNA synthetases as described and provided herein and encoded by the polynucleotides as described and provided herein. Said term may also be referred to herein as esterification of the cognate tRNA with an amino acid. The capability of polypeptides encoded by polynucleotides as described and provided herein to catalyze aminoacylation of cognate tRNAs with the respective amino acids may also be referred to herein as introducing or incorporating such amino acids into a polypeptide, which usually takes plays during the translation process of polypeptide synthesis. The aminoacyl tRNA synthetases according to the present invention are capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA. For example, a polypeptide encoded by the nucleotide sequence according to SEQ ID NO: 1 (pyrrolysine tRNA synthetase (PylRS) from Candidatus Methanomethylophilus alvus Mx1201 Ca) or 3 (PylRS from Candidatus Methanomethylophilus alvus Mx1201 Ca codon-optimized for E. coli) is capable of catalyzing aminoacylation of pyrrolysine (Pyl) with its cognate tRNA^Pyl (tRNAcu_A)- The polypeptide encoded by the nucleotide sequence according to SEQ ID NO: 1 has an amino acid sequence as shown in SEQ ID NO: 2, and the polypeptide encoded by the nucleotide sequence according to SEQ ID NO: 3 has an amino acid sequence as shown in SEQ ID NO: 4.

The term "cognate tRNA" as used in context with the present invention preferably means the tRNA which is charged or bonded with its corresponding amino acid to form an aminoacyl tRNA (also referred to herein as "aa tRNA"), a step which is preferably catalyzed by polypeptides encoded by polynucleotides as described and provided herein. "Cognate" in this sense is understood by the person skilled in the art and may particularly mean a pair of aaRS and tRNA carrying the corresponding anticodon corresponding to the codon for said amino acid on the mRNA, either base-by-base or by wobble base pairing as known in the art. That is, a given aaRS recognizes a specific cognate tRNA and loads (charges, etc. as described herein) it with a corresponding amino acid. For example, a tRNA_GAA (GAA being the anticodon of the tRNA) is charged with the corresponding amino acid phenylalanine (Phe) which is encoded on the mRNA by the codon UUC (or UUU). The tRNA_GAA is recognized by the specific aminoacyl tRNA synthetase for phenylalanine, phenylalanyl tRNA synthetase (PheRS), inter alia via its anticodon "GAA", and then charged by PheRS with phenylalanine to form the corresponding aminoacyl tRNA. The aminoacyl tRNA charged with Phe then recognizes the codon UUC (UUU) on the mRNA via its anticodon GAA (AAA) and phenylalanine can be introduced via peptide bonding into the growing polypeptide chain during translation process of polypeptide synthesis. Likewise, the tRNA_CuA is the tRNA for the corresponding non-canonical amino acid pyrrolysine (Pyl), encoded by UAG. Pairs of aminoacyl tRNA synthetases (aaRS) and its cognate tRNA which are charged by the aaRS with the corresponding amino acid are also referred to herein as "orthogonal pair". In one embodiment of the present invention, when being expressed in a host cell as described and provided herein, the aminoacyl tRNA synthetase described and provided herein is expressed together with its corresponding cognate tRNA as orthogonal pair. In one embodiment of the present invention, the tRNA corresponding to an aliphatic amino acid which is aminoacylated by the aaRS encoded by a polynucleotide as described and provided herein may be a tRNA^Pyl (tRNA_CuA), and the corresponding aliphatic amino acid may be Pyl, and the aaRS may be a pyrrolysyl tRNA synthetase (PylRS). In this context of the present invention, the orthogonal pair would be PylRS and its cognate tRNA^Pyl (tRNA_CuA)- Accordingly, in one embodiment of the present invention, when being expressed in a host cell as described and provided herein, the PylRS as described and provided herein is expressed in said host cell together with tRNA^Pyl (tRNAcu_A) as orthogonal pair. In a specific embodiment of the present invention, the PylRS and/or the corresponding orthogonal tRNA^Pyl (tRNA_CuA) may be derived from Methanomethylophilus alvus Mx1201 Ca.

In context with the present invention, the polynucleotide of the present invention encoding an aminoacyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA comprises a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence according to SEQ ID NO: 1 (pyrrolysine tRNA synthetase from Candidatus Methanomethylophilus alvus Mx1201 Ca) or 3 (codon-optimized sequence for E. coli as described herein), wherein said nucleotide sequence is codon-optimized to a selected host cell which is not Candidatus Methanomethylophilus alvus Mx1201 Ca.

It is preferred that the polynucleotide of the present invention encoding an aminoacyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA does not encode the polypeptides shown in the GenBank Entry with the accession no. CP002916, version CP002916.1 , preferably the sequence 990726..991574 with locus tag "TALC_01091 ", the UniProt Entry with the accession no. M9SC49 of 26 June 2013, the UniProt Entry with the accession no. A0A0W7TK26 of 16 March 2016 and/or the UniProt Entry with the accession no. M4YUJ0 of 29 May 2013. Furthermore, it is preferred that the polynucleotide of the present invention does not encompass the nucleotide sequence shown in SEQ ID NO. 13 of WO 2014/082773.

The term "codon-optimized", "codon-optimization", etc. as used herein is generally known in the art and inter alia relates to the fact that different organisms use different codons for the same amino acid in different frequencies ("codon usage" as known in the art). Due to different codon usage, it is possible that a gene from one organism may hardly or differently be expressed in another organism as the other organism uses one or more codons more often, more rarely or not at all and, thus, may have only few or no corresponding tRNAs for such codons and, consequently, suitable aminoacyl tRNA synthetases for such tRNAs. Accordingly, it may be required or at least advantageous to optimize the codons of a certain gene from one organism before transferring it to another organism in order to ensure comparable expression patterns. Such optimization usually takes place by substituting one or more bases within a nucleic acid sequence such as not to change its coded amino acid (i.e. silent mutation), but just to conform with the codon usage for a given amino acid codon with regard to the organism in which the nucleic acid molecule shall be expressed. For example, if in the original sequence of an aaRS as described and provided herein (e.g., PylRS derived from Candidatus Methanomethylophilus alvus Mx1201 Ca, SEQ ID NO: 1 ) the codon CTC for Leu and/or AGG for Arg is frequently contained, but the host cell (e.g., E. coli) in which the aaRS shall be expressed uses rather CTG for Leu and/or CGT for Arg, the codons in the nucleic acid sequence may be optimized accordingly in context with the present invention as also shown and exemplified herein (e.g., SEQ ID NO: 3: PylRS from Candidatus Methanomethylophilus alvus Mx1201 Ca codon-optimized for E. coli). As such, as used herein, a given polynucleotide sequence may be considered "codon- optimized" to a selected host cell if it contains one or more codons for a given amino acid which is used with the highest frequency for said amino acid in said host cell. In one embodiment of the present invention, a given polynucleotide sequence may be considered "codon-optimized" to a selected host cell if it contains one or more respective codons used with the highest usage frequency in said selected host cell for 1 , 2, 3 or all respective amino acids encoded by the polynucleotide. In a specific embodiment of the present invention, a given polynucleotide sequence may be considered "codon- optimized" to a selected host cell if it contains exclusively the respective codons with the highest usage frequency in the selected host cell for 1 , 2, 3 or all respective encoded amino acids, i.e. no codon with a lower usage frequency for 1 , 2, 3 or all respective encoded amino acids. Such codons with the highest frequency usage may be naturally contained in said polynucleotide or be introduced via suitable genetic modification tools known in the art (e.g., random or preferably site-specific mutations; PCR, restriction enzyme-based mutagenesis, CRISPR/Cas, etc). Codon-optimization may also and additionally refer to exchange of different stop codons. For example, if a given host cell expresses certain suppressor molecules for certain stop codons, the stop codon (e.g., amber, ochre or opal) may be adapted accordingly. Such process as defined herein above is referred to herein as "codon-optimization". For many though not all organisms, there are codon usage tables available which show the codon usage frequency for the respective host cell, i.e. which codons are used more often than others (and at which ratio). However, such codon usage tables are not available for all organisms (e.g., not for Candidates Methanomethylophilus alvus Mx1201 Ca). It is also one advantage of the present system that it is suitable to avoid interference with stop codons of the host cell while incorporating (preferably non-canonical) amino acids into polypeptides. For example, the aminoacyl tRNA synthetase described and provided herein (and encoded by the polynucleotide of the present invention) may be PylRS, which is able to aminoacylate a lysine derivative as described herein (e.g., pyrrolysine or boc-lysine) and its cognate tRNA^Pyl (tRNA_CuA)- As already mentioned, the anticodon CUA recognizes the amber stop codon (UAG) on the mRNA and, thus, acts as suppressor for the amber codon. In this context, when applied to host cells exhibiting a generally low level of amber stop codons within its respective genome, it is possible to avoid interference of incorporation of amino acids bound to their cognate tRNA (e.g., tRNA^Pyl) recognizing the amber stop codon.

As mentioned, the polynucleotide of the present invention encoding an aminoacyl tRNA synthetase (aaRS) capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described and also provided herein comprises a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence according to SEQ ID NO: 1 (pyrrolysine tRNA synthetase from Candidatus Methanomethylophilus alvus Mx1201 Ca) or 3 (codon-optimized sequence for E. coli as described herein), wherein said nucleotide sequence is codon-optimized to a selected host cell which is not Candidatus Methanomethylophilus alvus Mx1201 Ca. That is, such aaRS according to the present invention are capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described and exemplified herein. Likewise, the aminoacyl tRNA synthetase (aaRS) according to the present invention may have an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, provided it is capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA. The aaRS of the present invention may also be a fragment, e.g. be N- or C-terminally or internally deleted compared to the amino acid sequence according to SEQ ID NO: 2, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deletions from the N- or C-terminus or internally of the polypeptide, provided such fragment is capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described herein. Also, the aaRS as described and provided herein may encompass 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions or substitutions compared to the amino acid sequence of SEQ ID NO: 2, preferably it may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative or highly conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2. As used herein, "silent" mutations mean base substitutions within a nucleic acid sequence which do not change the amino acid sequence encoded by the nucleic acid sequence. "Conservative" substitutions mean substitutions as listed as "Exemplary Substitutions" in Table I below. "Highly conservative" substitutions as used herein mean substitutions as shown under the heading "Preferred Substitutions" in Table I below.

The term "position" when used in accordance with the present invention means the position of an amino acid within an amino acid sequence depicted herein. The term "corresponding" in this context also includes that a position is not only determined by the number of the preceding nucleotides/amino acids. The level of identity between two or more sequences (e.g., nucleic acid sequences or amino acid sequences) can be easily determined by methods known in the art, e.g., by BLAST analysis. Generally, in context with the present invention, if two sequences (e.g., polynucleotide sequences or amino acid sequences) to be compared by, e.g., sequence comparisons differ in identity, then the term "identity" may refer to the shorter sequence and that part of the longer sequence that matches said shorter sequence. Therefore, when the sequences which are compared do not have the same length, the degree of identity may preferably either refer to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence or to the percentage of nucleotides in the longer sequence which are identical to nucleotide sequence in the shorter sequence. In this context, the skilled person is readily in the position to determine that part of a longer sequence that matches the shorter sequence. Furthermore, as used herein, identity levels of nucleic acid sequences or amino acid sequences may refer to the entire length of the respective sequence and is preferably assessed pair-wise, wherein each gap is to be counted as one mismatch. These definitions for sequence comparisons (e.g., establishment of "identity" values) are to be applied for all sequences described and disclosed herein.

TABLE I Amino Acid Substitutions

Moreover, the term "identity" as used herein means that there is a functional and/or structural equivalence between the corresponding sequences. Nucleic acid/amino acid sequences having the given identity levels to the herein-described particular nucleic acid/amino acid sequences may represent derivatives/variants of these sequences which, preferably, have the same biological function. They may be either naturally occurring variations, for instance sequences from other varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques. Deviations from the above-described nucleic acid sequences may have been produced, e.g., by deletion, substitution, addition, insertion and/or recombination. The term "addition refers to adding at least one nucleic acid residue/amino acid to the end of the given sequence, whereas "insertion" refers to inserting at least one nucleic acid residue/amino acid within a given sequence. The term "deletion" refers to deleting or removal of at least one nucleic acid residue or amino acid residue in a given sequence. The term "substitution" refers to the replacement of at least one nucleic acid residue/amino acid residue in a given sequence. Again, these definitions as used here apply, mutatis mutandis, for all sequences provided and described herein.

Generally, as used herein, the terms ..polynucleotide" and ..nucleic acid" or ..nucleic acid molecule" are to be construed synonymously. Generally, nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules. Furthermore, the term "nucleic acid molecule" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 552571 1 , US 471 1955, US 5792608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332 - 4339). Said polynucleotide sequence may be in the form of a vector, plasmid or of viral DNA or RNA. Also described herein are nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present invention. Particularly, such a fragment is a functional fragment. Examples for such functional fragments are nucleic acid molecules which can serve as primers.

The term "hybridization" or "hybridizes" as used herein in context of nucleic acid molecules/DNA sequences may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N. Y. (2001 ); Current Protocols in Molecular Biology, Update May 9, 2012, Print ISSN: 1934-3639, Online ISSN: 1934-3647; Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N. Y. (1989), or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as 0.1 x SSC, 0.1 % SDS at 65 °C. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6 x SSC, 1 % SDS at 65 °C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. In accordance to the invention described herein, low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1 % SDS at 65 °C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.

Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may represent nucleic acid molecules which code for a functional aaRS as described herein or a functional fragment thereof which can serve as a primer. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands. The term "hybridizing sequences" preferably refers to sequences which display a sequence identity of at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%. more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% more preferably at least 99%, more preferably at least 99,5%, and most preferably 100% identity with a nucleic acid sequence as described herein encoding an aaRS as described and provided herein.

In one embodiment of the present invention, the nucleotide sequence comprised by the polynucleotide of the present invention encoding an aminoacyl tRNA synthetase (aaRS) capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described and also provided herein which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence according to SEQ ID NO: 1 or 3 comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, or 17 silent mutations where CTC (Leu)-codons are replaced by CTG (Leu)-codons, AGG (Arg)-codons are replaced by CGT (Arg)-codons, and/or the TGA (opal stop codon) is replaced by TAA (ochre stop codon). Any one or more of these silent mutations may serve as codon-optimization of the polynucleotide to any host cell which has a higher codon usage of CTG for Leu compared to CTC for Leu, and/or higher codon usage of CGT for Arg compared to AGG for Arg. A non-exhaustive example for a host cell using more CTG than CTC for Leu and more CGT than AGG for Arg comprises E. coli. For example, in context with the present invention, said nucleotide sequence may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, or 17 of any one of the following substitutions or pairs of substitutions a) to q), compared to the nucleotide sequence of SEQ ID NO: 1 :

a) C39G

b) C87G

c) A94C and G96T

d) A343C and G345T

e) C351 G

f) A352C and G354T

g) A388C and G390T

h) A397C and G399T

i) A448C and G450T

j) C501 G

k) C579G

1) C594G

m) C687G

n) C738G

o) A757C and G759T

P) C807G, and/or

q) G827A,

provided said polynucleotide encodes an aminoacyl tRNA synthetase (aaRS) capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described herein.

In one embodiment of the present invention, the nucleic acid sequence comprises substitution q) and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 substitutions or pairs of substitutions of any one of a) to p).

In one embodiment of the present invention, the nucleotide sequence comprised by the polynucleotide of the present invention encoding an aminoacyl tRNA synthetase (aaRS) capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described and also provided herein which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence according to SEQ ID NO: 1 or 3 comprises only nucleotide deviations compared to nucleotides 1 to 825 of SEQ ID NO: 1 which only result in no, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more silent mutations, or conservative or highly conservative amino acid substitutions when being expressed to a polypeptide.

In one embodiment of the present invention, the amino acid which is aminoacylated with its corresponding tRNA by the aaRS as described and provided herein may be a lysine or, preferably, a lysine derivative. In one embodiment of the present invention, such lysine derivative may be selected from the group consisting of pyrrolysine (Pyl), boc- lysine (A/_a-(fert-Butoxycarbonyl)-L-lysine), alloc-lysine (N₆-Alloc-L-lysine), azide-lysine, 2- N,6-N-Bis(2,3-dihydroxy-N-benzoyl)-L-serine, 2-N,6-N-Bis(2,3-dihydroxy-N-benzoyl)-L- serine amide, 3-hydroxylysine, /V-benzoylglycyl-A^-^-hydroxy^-iS-methylquinoxalin^- yl)ethyl]lysine, A/-benzoylglycyl-A/⁶-[2-hydroxy-3-(quinoxalin-2-yl)propyl]lysine, N- hippuryl-A^-icarboxymethylJIysine, A/⁶-(2,4-dinitrophenyl)lysine, ^-(2- carboxyethyl)lysine, A^-acetonyllysine, /S^-carbamoylmethyllysine, A^-methyllysine, hydroxylysine, isodesmosine, ornithine derivatives such as 2-amino-5-(prop-2- ynoylamino)pentanoic acid (5-(prop-2-ynoylamino)ornithine), and 2-amino-5- [(azidoacetyl)amino]pentanoic acid. In a specific embodiment, the lysine derivative is pyrrolysine or boc-lysine.

In one embodiment of the present invention, the polynucleotide provided and described herein comprises or consists of a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence according to SEQ ID NO: 3 (PylRS derived from Mma, codon-optimized for expression in E. coli), provided the polypeptide encoded by said polynucleotide is an aminoacyl tRNA synthetase (aaRS) capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described and provided herein.

The present invention also relates to a vector comprising a polynucleotide described and provided in accordance with the present invention. That is, the present invention also relates to a vector comprising a polynucleotide as described and provided herein encoding an aminoacyl tRNA synthetase (aaRS) capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described and also provided herein, said polynucleotide comprising a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence according to SEQ ID NO: 1 or 3, wherein said nucleotide sequence is codon-optimized to a selected host cell which is not Candidates Methanomethylophilus alvus Mx1201 Ca. The present invention also relates to a vector comprising a polynucleotide encoding a tRNA which corresponds to the amino acid with which said tRNA is aminoacylated by the aminoacyl tRNA synthetase (aaRS) as described and provided herein. For example, if the amino acid which is charged to the corresponding tRNA by the aaRS (e.g., PylRS, for example the PylRS from Mma according to SEQ ID NO: 2) as described herein is a pyrrolysine or boc-lysine, or AllocK, or AzideK, or another pyrrolysine derivative as described herein, the tRNA may be tRNA^Pyl (e.g., MmatRNA^Pyl according to SEQ ID NO: 5 to have the orthogonal Mma-pair (MmaOP), or /WmtRNA^Pyl derived from Mm to have a hybrid pair). In one embodiment of the present invention, the tRNA may be encoded by a polynucleotide comprising a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence according to SEQ ID NO: 5 (tRNA^Pyl derived from Mma) tRNA^Pyl derived from Mm, wherein said nucleotide sequence being at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence according to SEQ ID NO: 5 may be codon-optimized to a selected host cell, and wherein said host cell is preferably not Candidatus Methanomethylophilus alvus Mx1201 Ca. The MmatRNAcu_A cassette comprising further a promoter and a terminator region is shown in SEQ ID NO: 6 and is also a tRNA which may be encoded by a polynucleotide which may be codon-optimized according to this invention. The vector may comprise such polynucleotide encoding a tRNA as described above in addition to the polynucleotide encoding the aaRS as described further above, wherein both polynucleotides may be under the control of the same or different promoters. In context with the present invention, such tRNAs may also comprise mutated or engineered tRNAs which exhibit altered (e.g., higher) binding affinity or substrate specificity to its corresponding aaRS or altered (e.g., higher) to its corresponding amino acid, or altered anticodon-codon binding behaviors. Such altered anticodon-codon binding behaviors may be in such a way that specific codons are recognized more specifically by the tRNAs anticodon (i.e. higher base-to-base binding specificity, less wobble-base pairing abilities), e.g., either by altering the anticodon itself or by adapting the special structure of the tRNA such as to alter the binding behavior. Methods for engineering tRNAs in this context are known in the art and comprise those as described in, e.g., Liu et al., PNAS (1997), 94: 10092- 10097; Wang and Schultz, Chem Biol (2001 ), 8: 883-890; and Maranhao and Ellington, ACS Synth Biol., Evolving Orthogonal Suppressor tRNAs To Incorporate Modified Amino Acids, DOI 10.1021 /acssynbio.6b00145. Also, in context with the present invention, the tRNAs may be engineered such as to enhance the affinity of aminoacylated tRNA to the EF (elongation factor) Tu (cf., e.g., Schrader et al., PNAS (201 1 ), 108(13): 5215-5220).

The term "vector" as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In one embodiment of the present invention, the vectors are suitable for the transformation, transduction and/or transfection of host cells as described herein, e.g., prokaryotic cells (e.g., (eu)bacteria, archaea), eukaryotic cells (e.g., mammalian cells, insect cells) fungal cells, yeast, and the like. Examples of bacterial host cells in context with the present invention comprise Gram negative and Gram positive cells. Specific examples for suitable host cells may comprise inter alia E. coli, Mycoplasma capricolum, SF9 cells, CHO, C. elegans cell, S. cerevisiae, Schizosaccharmyces pombe, Micrococcus luteus, Pichia pastoris (today also known as Komagataella pastoris or Komagataella phaffii), plant cells, and Bombyx mori. In one embodiment of the present invention, said vectors are suitable for stable transformation of the host cells, for example to express the aaRS and/or the tRNA as described and provided herein.

Accordingly, in one aspect of the invention, the vector as provided is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is preferably at least one restriction site or a polylinker which enables the insertion of a nucleic acid sequence/molecule desired to be expressed. It is to be understood that when the vector provided herein is generated by taking advantage of an expression vector known in the prior art that already comprises a promoter suitable to be employed in context of this invention, for example expression of an aaRS and/or tRNA as described herein. The nucleic acid construct is preferably inserted into that vector in a manner the resulting vector comprises only one promoter suitable to be employed in context of this invention. The skilled person knows how such insertion can be put into practice. For example, the promoter can be excised either from the nucleic acid construct or from the expression vector prior to ligation. In one embodiment of the present invention, the vector is able to integrate into the host cell genome. The vector may be any vector suitable for the respective host cell, preferably an expression vector. The vector may comprise the polynucleotide encoding an aminoacyl tRNA synthetase (aaRS) capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA as described and also provided herein, and/or the cognate tRNA as described and provided herein. A non-limiting example of the vector of the present invention may comprise pSCS (see, e.g., Figure 1 ) comprising the polynucleotide in context of the present invention.

The present invention also relates to host cells comprising a polynucleotide as described and provided herein comprising a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence according to SEQ ID NO: 1 or 3, wherein said polynucleotide encodes an amino acyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic or aliphatic amino acid to form an aminoacyl-tRNA, wherein said host cell is not Methanomethylophilus alvus Mx1201 Ca. In one embodiment of the present invention, said polynucleotide comprised by said host cell is codon-optimized for said host cell as described herein. The present invention also relates to a host cell which comprises a polynucleotide encoding a cognate tRNA corresponding to the amino acid with which said tRNA is aminoacylated by the aminoacyl tRNA synthetase (aaRS) as described and provided herein, or a vector containing said polynucleotide as also described herein. The present invention also relates to host cells comprising a vector comprising the polynucleotide encoding an aaRS as described and provided herein, and/or the polynucleotide encoding a tRNA as described and provided herein. In one embodiment of the present invention, said host cell of the present invention is able to stably express the polynucleotide and/or vector. In one embodiment of the present invention, the host cell comprises a polynucleotide encoding the aaRS as described and provided herein, and the corresponding tRNA as also described herein, for example as orthogonal pair (e.g., MmaPyIRS with MmatRNA^Pyl) or as hybrid pair (e.g., MmaPyIRS with a tRNA^Pyl derived from another organism than Mma, such as tRNA^Pyl derived from Mm, or vice versa). In this context, to obtain a host cell comprising a hybrid pair of aaRS and tRNA, it is also envisaged in context with the present invention to combine the PylRS derived from another organism than Mma, e.g., PylRS derived from Mm (/WmPyIRS), with a tRNA of another organism, e.g., natRNA^Pyl (e.g., as shown in SEQ ID NO: 5). That is, the present invention also relates to a host cell comprising a polynucleotide encoding MmPylRS and a polynucleotide encoding MmatRNA^Pyl (e.g., as shown in SEQ ID NO: 5), i.e. a hybrid pair. Such polynucleotides may be localized on one polynucleotide, e.g., one vector, or on different polynucleotides, e.g. vectors. The present invention also relates to such vectors encoding ΛfmPylRS and/or /WmatRNA^Pyl (e.g., as shown in SEQ ID NO: 5). Such MmPyIRS and/or tRNA (e.g., /W/natRNA^Pyl) may also further be codon-optimized for specific host cells as described and exemplified herein.

In one embodiment of the present invention, the host cell of the present invention may comprise (i) a polynucleotide encoding an aminoacyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aliphatic or aromatic amino acid to form an aminoacyl-tRNA, said polynucleotide comprising a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence according to SEQ ID NO: 1 (pyrrolysine tRNA synthetase from Candidatus Methanomethylophilus alvus Mx1201 Ca) or 3 (codon- optimized sequence for E. coli as described herein), wherein said nucleotide sequence may be codon-optimized to a selected host cell, and wherein said host cell is preferably not Candidatus Methanomethylophilus alvus Mx1201 Ca, and/or (ii) a polynucleotide encoding a corresponding tRNA, said polynucleotide comprising a nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence according to SEQ ID NO: 5 (tRNA^Pyl derived from Mma) or to tRNA^Pyl derived from Mm, wherein said nucleotide sequence may be codon-optimized to a selected host cell, and wherein said host cell is preferably not Candidatus Methanomethylophilus alvus Mx1201 Ca.

The host cell of the present invention may generally be any host cell, preferably being capable of stably expressing a polynucleotide encoding an aaRS and/or a tRNA as described and provided herein. Such host cells may comprise, inter alia, e.g., prokaryotic cells (e.g., (eu)bacteria, archaea), eukaryotic cells (e.g., mammalian cells, insect cells) fungal cells, yeast, or entire organisms (e.g., Bombyx mori) and the like. In one embodiment of the present invention, the host cell is not archaea. Examples of bacterial host cells in context with the present invention comprise Gram negative and Gram positive cells. Specific examples for suitable host cells may comprise inter alia E. coli, Mycoplasma capricolum, CHO, SF9 cells, C. elegans cell, S. cerevisiae, Schizosaccharmyces pombe, Micrococcus luteus, Pichia pastoris (today also known as Komagataella pastoris or Komagataella phaffii), plant cells, and Bombyx mori.

The present invention further relates to a polypeptide encoded by a polynucleotide as described and provided herein, particularly a polynucleotide encoding an aaRS and/or a tRNA as described and provided herein.

That is, the present invention also relates to an aminoacyl tRNA synthetase (aaRS) as described and provided herein, and to a tRNA as described and provided herein. In one embodiment of the present invention, the aminoacyl tRNA synthetase may be a pyrrolysine tRNA synthetase (PylRS), for example the PylRS derived from Candidatus Methanomethylophilus alvus Mx1201 Ca. Such PylRS may preferably be capable of catalyzing the aminoacylation of lysin or a lysin derivative (e.g., pyrrolysine or boc- lysine) with its cognate tRNA (e.g., tRNA^Pyl (tRNA_CUA))- For example, such PylRS may have an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 4. As already mentioned and described, the present invention also relates to polynucleotides encoding such aaRS as well as to vectors comprising such polynucleotides and host cells comprising such polynucleotides and/or vectors.

The present invention also relates to a cognate tRNA which corresponds to an amino acid with which it is aminoacylated by an aaRS of the present invention. In one embodiment of the present invention, such tRNA may be at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the MmatRNA_CUA as described herein (tRNA^Pyl derived from Candidatus Methanomethylophilus alvus Mx1201 Ca). As already mentioned and described, the present invention also relates to polynucleotides encoding such tRNA as well as to vectors comprising such polynucleotides and host cells comprising such polynucleotides and/or vectors. The present invention also relates to a composition comprising an orthogonal pair of an aminoacyl tRNA synthetase as described and provided herein and its cognate tRNA as described and provided herein. The present invention further relates to the use of a polynucleotide encoding an aminoacyl tRNA synthetase (aaRS) as described and provided herein, the use of a vector comprising such polynucleotide, the use of a host cell comprising such vector and/or polynucleotide, the use of a polypeptide encoded by such polynucleotide or an aminoacyl tRNA synthetase as described and provided herein, to catalyze the introduction of an aliphatic or aromatic amino acid as described herein into a polypeptide. As known in the art, such introduction usually takes place during the translation process of polypeptide biosynthesis.

The present invention further relates to the use of a polynucleotide encoding a tRNA as described and provided herein, the use of a vector comprising such polynucleotide, the use of a host cell comprising such vector and/or polynucleotide, the use of a polypeptide encoded by such polynucleotide or an aminoacyl tRNA synthetase as described and provided herein, to catalyze the introduction of an aliphatic or aromatic amino acid as described herein into a polypeptide. As known in the art, such introduction usually takes place during the translation process of polypeptide biosynthesis.

The present invention further relates to the use of an orthogonal pair of aminoacyl tRNA synthetase and its cognate tRNA as described and provided herein to catalyze the introduction of an aliphatic or aromatic amino acid as described herein into a polypeptide. As known in the art, such introduction usually takes place during the translation process of polypeptide biosynthesis.

The present invention also relates to a method of incorporating an aliphatic or aromatic amino acid as described herein into a polypeptide, comprising the following steps:

(i) expressing a polynucleotide encoding an aminoacyl tRNA synthetase as described and provided herein, and

(ii) expressing a polynucleotide encoding a tRNA (which is cognate to the aminoacyl tRNA synthetase as described and provided herein),

said aminoacyl tRNA synthesis and tRNA being an orthogonal pair. The embodiments which characterize the present invention are described herein, shown in the Figures, illustrated in the Examples, and reflected in the claims. It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term". The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

The Figures show:

Expression vectors pSCS+ comprising PylRS and its cognate tRNAcu_A and pET21a comprising MmaPyIRS with lacl

(a) Plasmid map of the suppressor plasmid (pSCS+). The plasmid carried the o-pair coding for the PylRS (blue arrow) and the suppressor tRNA_CUA (red arrow). The PylRS was expressed under the control of the inducible arabinose promoter {PBAD) and the cognate tRNAcu_A under the control of the constitutive proK promoter (PproK). An IPTG-inducible pT5//acO promoter regulated the expression of the eGFP reporter gene carrying an in-frame amber stop codon (TAG) at positon 40 (eGFP[Y40am], green arrow). Since the plasmid was missing the lacl repressor, the eGFP is constitutively expressed. The plasmid also carried the following elements: proK terminator; neokan, kanamycin resistance marker; p15a, medium-copy origin of replication; rrnBTI, rrnBT2, rrnC and lamda to, terminators. (b) Plasmid map of the IPTG-inducible pET21 a(+) expression construct of MmaPylRS-his₆.

Figure 2 Orthogonality of MmaPylRS//WmatRNAc_UA in E. coli

/WmaPylRS/iWmatRNAcuA pair was used to introduce the Pyl analog BocK into eGFP[Y40am]. To prove its orthogonality in E. coli, the AtfmaPylRS was expressed without a tRNA_CuA (-)■ Orthogonal pair of Mm was used as comparison and cells were supplemented with 10 mM NaPi pH 8.0 (-) to detect the background suppression in the absence of the Pyl analog (fidelity control). Absolute fluorescence units (A_ex 488 nm; A_em 508 nm) were recorded 4 h (white) and 24 h (black) after induction of the PylRS with 0.2% arabinose. Fluorescence units were correlated to cell density ( 488/508/D600) and the average of eight technical replicates is shown. Error bars indicate standard deviations.

Figure 3 /WmaPylRS-his₆ expression in active form in E. coli

(a) BocK was incorporated into eGFP[Y40am] to assess the functionality of Λ imaPylRS carrying a C-terminal hexhistidine tag. Cells were supplemented with 10 mM NaPi pH 8.0 to detect the background suppression (fidelity control). The eGFP fluorescence (F488 508, A_ex 488 nm; A_em 508 nm) was recorded 4 h (white) and 24 h (black) after induction of the reporter protein. The average of eight technical replicates is shown, error bars indicate standard deviations.

(b) eGFP[Y40BocK] (calculated M_w 28 kDa; closed arrow) was purified, as well as MmaPylRS-his₆ (calculated M_w 32 kDa; open arrow) from crude lysates of cultures lacking BocK (-) or supplemented with 5 mM BocK (+). The Af/naPylRS-his₆ band was excised and the sequence was confirmed by in-gel mass analysis (not shown). M, molecular size marker; S, soluble protein fraction; I, insoluble protein fraction; E, eluate. 5 μg of of total protein were loaded per lane. Coomassie stained 4-12% Bis-Tris SDS gels are shown. Figure 4 Differences in ncAA incorporation between Λί/na Pyl RS and AfmPylRS

Pyl analog BocK was used to suppress the in-frame amber stop codon of eGFP[Y40am]. Cells were supplemented with 10 mM NaPi pH 8.0 to detect the background suppression (fidelity control). The eGFP fluorescence (F _Ba/508, A_ex 488 nm; A_em 508 nm) was recorded 4 h (white) and 24 h (black) after induction of the PylRS with 0.2% arabinose. Fluorescence units were correlated to cell density (F ₈₈ 508/D₆o₀) and the average of eight technical replicates is shown. Error bars indicate standard deviations.

Figure 5 Mass analysis eGFP[Y40BocK]

The monoisotopic masses of eGFP[Y40BocK] produced by cells expressing the MmaOP are shown (black sticks). Protein species missing a few N- and C-terminal amino acids were detected. The peak with the largest mass corresponds to the less truncated species (for details see Table II). Starting from this mass further N-terminal (dotted horizontal line) and C-terminal (full horizontal line) truncated species of the eGFP[Y40BocK] were idientified. The missing amino acids are indicated by the 1 -letter code. Exact monoisotopic masses are summarized in Table II and amino acid sequence of eGFP[Y40am] is shown in SEQ ID NO: 8. All species in the indicated mass window with a relative abundance >10% are shown. Grey sticks indicate unidentified fragments.

Figure 6 SCS experiment with BocK (white), AllocK (grey) and AzideK (black) using the MmaOP does not affect E. co/i growth

D₆₀₀ was measured before induction, 4 and 24 hours after induction of the aaRS expression. Red lines indicate the timepoint of arabinose addition. Two independent biological values are shown (circles and squares).

Figure 7 Mass analysis The monoisotopic masses of eGFP[Y40BocK] (a), eGFP[Y40AllocK] (b) and eGFP[Y40AzideK] (c) produced by cells expressing the MmaOP are shown (black sticks). Protein species missing a few N- and C-terminal amino acids were detected. The peak with the largest mass corresponds to the eGFP[Y40ncAA]_trunc species missing the amino acids

MRSHHHHHH at the N-terminus and LYK at the C-terminus (for details see Table III). Comparison of this truncated species confirms the incorporation of the desired ncAA since the mass differences of the species correspond to mass differences of the incorporated ncAAs: AeGFP[Y40BocK]_trunc (a; Mw 26709) vs. eGFP[Y40AllocK]_trunc (b;

Mw 26693) = 16 (calculated 16.04); AeGFP[Y40BocK]_trunc (a; Mw 26709) vs. eGFP[Y40AzideK]_trunc (c; Mw 26722) = 13 (calculated 12.96); AeGFP[Y40AllocK]_trunc (b; Mw 26693) vs. eGFP[Y40AzideK]_tmnc (c; Mw 26722) = 29 (cal: 29.00). Starting from this mass, further N-terminal (dotted horizontal line) and C-terminal (full horizontal line) truncated species of the eGFP[Y40ncAA] were identified. The missing amino acids are indicated by the 1 -letter code. Exact monoisotopic masses are summarized in Table III and amino acid sequence of eGFP[Y40am] is shown in SEQ ID NO: 8. All species in the indicated mass window with a relative abundance >10% are shown. Grey sticks indicate not identified fragments.

The following sequences are described and provided herein: SEQ ID NO: 1

DNA Candidates Methanomethylophilus alvus Mx1201 Ca

MmaPyIRS

atgacggtaaagtatacggatgcacagatacagcgcctcagggaatacggcaacgggacctatgagcagaaggtcttc gaggacctcgcatcgagggatgctgccttctccaaggagatgtccgtcgcctctaccgacaacgagaagaagatcaagg ggatgatcgccaatccgtcccgtcatggattgacccagctgatgaacgacatcgccgacgcattggtcgccgagggtttcat cgaggtccgtacgcccatattcatatcgaaggatgcgctggcacgtatgaccatcaccgaggacaagccccttttcaagca ggtcttctggatcgacgagaaaagggcgctcaggcctatgctggcacctaacctttattccgtcatgagggacctgaggga ccatacggacggtccggtgaagatcttcgagatgggttcctgcttcaggaaggagtcccacagcgggatgcatctggagg agttcaccatgctgaacctcgtggacatgggtccccgcggagacgccacggaggtcctgaagaactacatatcggtcgtg atgaaggcggccggtctcccggactacgacctcgtacaggaggagtccgacgtatacaaggagaccatagacgtggag atcaacggtcaggaggtctgttccgcagccgtcggtccacactatctcgatgcggcccacgatgtccacgagccttggtcc ggagcgggattcggtctcgaacgcctgctgaccatcagggagaagtacagcaccgtgaagaagggaggagccagcat cagctacctcaacggtgcgaagatcaactga

SEQ ID NO: 2

Protein Candidatus Methanomethylophilus alvus Mx1201 Ca

MmaPyIRS

MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANP SR

HGLTQLMNDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPM

LAPNLYSVMRDLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDAT

EVL

KNYISVVMKAAGLPDYDLVQEESDVYKETIDVEINGQEVCSAAVGPHYLDAAHDVHEP WS

GAGFGLERLLTIREKYSTVKKGGASISYLNGAKIN*

SEQ ID NO: 3

DNA artificial

MmaPyIRS codon-optimized for E. coli

atgacggtaaagtatacggatgcacagatacagcgcctGCGTgaatacggcaacgggacctatgagcagaaggtctt cgaggacctggcatcgcgtgatgctgccttctccaaggagatgtccgtcgcctctaccgacaacgagaagaagatcaag gggatgatcgccaatccgtcccgtcatggattgacccagctgatgaacgacatcgccgacgcattggtcgccgagggtttc atcgaggtccgtacgcccatattcatatcgaaggatgcgctggcacgtatgaccatcaccgaggacaagccccttttcaag caggtcttctggatcgacgagaaacgtgcgctgcgtcctatgctggcacctaacctttattccgtcatgcgtgacctgcgtgac catacggacggtccggtgaagatcttcgagatgggttcctgcttccgtaaggagtcccacagcgggatgcatctggaggag ttcaccatgctgaacctggtggacatgggtccccgcggagacgccacggaggtcctgaagaactacatatcggtcgtgat gaaggcggccggtctgccggactacgacctggtacaggaggagtccgacgtatacaaggagaccatagacgtggaga tcaacggtcaggaggtctgttccgcagccgtcggtccacactatctggatgcggcccacgatgtccacgagccttggtccg gagcgggattcggtctggaacgcctgctgaccatccgtgagaagtacagcaccgtgaagaagggaggagccagcatca gctacctgaacggtgcgaagatcaactaa

SEQ ID NO: 4

Protein artificial MmaPyIRS codon-optimized for E. coli

MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANP SR

HGLTQLMNDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPM LAPNLYSVMRDLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDAT EVL

KNYISVVMKAAGLPDYDLVQEESDVYKETIDVEINGQEVCSAAVGPHYLDAAHDVHEP WS

GAGFGLERLLTIREKYSTVKKGGASISYLNGAKIN^*

SEQ ID NO: 5

DNA Candidatus Methanomethylophilus alvus Mx1201 Ca

MmatRNAcuA

GGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCCAGCGGGGTTCGAC GCCCCGGTCTCTCGCCAA

SEQ ID NO: 6

DNA artificial

MmatRNAcuA cassette

The sequence of /WmatRNA_CUA (bold) is flanked by the proK promoter (italic) and terminator.

AGGCATTTTGCTATTAAGGGATTGACGAGGGCGTATCTGCGCAGTAAGATGCGCCC

CGC/47TGGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCCAGCGGG

GTTCGACGCCCCGGTCTCTCGCCAAATTCGAAAAGCCTGCTCAACGAGCAGGCTT TTTG

SEQ ID NO: 7

DNA artificial

MmaPylRS-hiSg

The sequence of the GS-linker and the C-terminal hexahistidine tag is shown in capital letters

atgacggtaaagtatacggatgcacagatacagcgcctGCGTgaatacggcaacgggacctatgagcagaaggtctt cgaggacctggcatcgcgtgatgctgccttctccaaggagatgtccgtcgcctctaccgacaacgagaagaagatcaag gggatgatcgccaatccgtcccgtcatggattgacccagctgatgaacgacatcgccgacgcattggtcgccgagggtttc atcgaggtccgtacgcccatattcatatcgaaggatgcgctggcacgtatgaccatcaccgaggacaagccccttttcaag caggtcttctggatcgacgagaaacgtgcgctgcgtcctatgctggcacctaacctttattccgtcatgcgtgacctgcgtgac catacggacggtccggtgaagatcttcgagatgggttcctgcttccgtaaggagtcccacagcgggatgcatctggaggag ttcaccatgctgaacctggtggacatgggtccccgcggagacgccacggaggtcctgaagaactacatatcggtcgtgat gaaggcggccggtctgccggactacgacctggtacaggaggagtccgacgtatacaaggagaccatagacgtggaga tcaacggtcaggaggtctgttccgcagccgtcggtccacactatctggatgcggcccacgatgtccacgagccttggtccg gagcgggattcggtctggaacgcctgctgaccatccgtgagaagtacagcaccgtgaagaagggaggagccagcatca gctacctgaacggtgcgaagatcaacGGTTCTCATCACCATCACCATCACTAA

SEQ ID NO: 8

Protein artificial

eGFP[Y40am]

MRSHHHHHHGSMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATXGKLTLK FICTTGKLPVPWPTLN TTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK^* Primers used herein:

Underlined sequences indicate the priming sequence, the GS-linker is shown in italic and the hexahistidine tag in bold

SEQ ID NO: 9

DNA artificial

Primer BPp1496

GTTG GAG CTCG AG G CCTCGTCG ACTTAAAG AG G AG AAATTAACTATG SEQ ID NO: 10

DNA artificial

Primer BPp1497

TTATTTGATGCCTGATGGTCGACTTAGTTGATCTTCGCACC

SEQ ID NO: 1 1

DNA artificial Primer BPp81 1

ATTTAAATTCTAG AG ACCCATTAATCTG C AG CTTG CAG G CATTTTG CTATTAAG G G A TTG SEQ ID NO: 12

DNA artificial

Primer BPp812

CCCGAAAAGTGCCACCTGCATCGATTTACTGCAGGTAAAAAAAGCCTGCTCGTTGA G

SEQ ID NO: 13

DNA artificial

Primer BPp1498

TTATTTGATGCCTGATGGAAGCCTTTAGTGATGGTGATGGTGATG/4GA4CCGTTGA TCTTCGCACCGTTCAG

The invention is further illustrated by the following examples, however, without being limited to the example or by any specific embodiment of the examples. Examples

Example 1 : Preparing Codon-optimized PylRS

The CTC and AGG codons of MmaPyIRS (SEQ ID NO: 1 ) were exchanged in the coding sequence of MmaPyIRS to CTG and CGT (SEQ ID NO: 3) to adapt it to the codon usage of E. coli. The codon harmonization was made since CTC and AGG are frequent codons for Leu and Arg in Mma while they are rare in E. coli. Further codon optimizations were not possible since a full codon usage table was not available for Mma. The nucleotide sequence for tRNA_CUA^Pyl (tRNA_CUA; pylT) was taken from Mma (SEQ ID NO: 5). Example 2: Orthogonality of MmaPylRS/MmatRNA_CUA in E. coli

To verify the functionality of the MmaPylRS/MmatRNAcuA pair, the pSCS+ screening plasmid was used (Figure 1 ). On pSCS+, a single copy of the target PylRS was introduced at the Sail site and expressed from the arabinose-inducible pBAD promoter. The sequence encoding the tRNAcu_A was inserted at the Pstl site and was flanked by an upstream proK promoter and a downstream proK terminator . pSCS+ encodes the eGFP reporter carrying an in-frame amber (TAG) codon at positon Y40 (eGFP[Y40am]) to validate the performance of a PylRS and its cognate tRNA_CuA- Y40 is a 'permissive site', that is the insertion of amino acid analogs at this position does not interfere with eGFP fluorescence. A functional PylRS/tRNA_CUA pair can incorporate Pyl or a non-canonical analog at the TAG codon and thus leads to the suppression of translation at this position and the expression of full length eGFP. Consequently, the fluorescence read-out directly correlates with a functional PylRS/tRNA_CUA pair. BL21 cells harboring the pSCS+ plasmid were cultivated in LB medium in the presence or absence of the Pyl analog N_E-Boc-L-lysine (BocK) for 4 and 24 hours. BocK is a substrate for /WmPyIRS and is commercially available. Cells expressing the MmaPylRs/ZWmatRNAcuA pair from pSCS+ showed eGFP fluorescence (A_em 508 nm; λ_ΘΧ 488 nm) when BocK was added to the culture medium. The fluorescence signal increased with time. Without the supplementation of BocK a significant increase in the eGFP fluorescence was not detected (Figure 2). The fluorescence readout in the presence of BocK provided evidence that the MmaPylRS was functionally expressed in E. coli together with its cognate tRNA_CuA- The enzyme accepted BocK as a substrate, which was not expected due to the low sequence homology between Af/naPylRS and MmPyIRS, which accepts BocK. Moreover, even less expectable, M/naPylRS incorporated BocK much more efficiently than /WmPyIRS (Figure 2). Insertion of BocK was confirmed in response to the in-frame amber stop codon of eGFP[Y40am] by mass analysis (Figure 5 and Table II).

Table II: Mass Analysis 1

Calculated monoisotopic masses of the main species of eGFP[Y40am] variants with the proposed N and C-terminal truncations are compared to the experimentally found masses; eGFP chromophore formation was integrated.

eGFP[Y40BocK]

o-pair N_term Cterm m3ss_ca|_CU|_at_ed masSf_0und Amass

Mma MRSHHHHHH - 27,1 13.06 27,1 13.77 0/71

(SEQ ID NO: 14) Mma MRSHHHHHHGS 26,969.01 26,969.73 0.72

(SEQ ID NO: 15)

Mma MRSHHHHHHGSM 26,837.97 26,838.68 0.29

(SEQ ID NO: 16)

Mma MRSHHHHHHGSMV LYK 26,335.21 26,334.42 0.79

(SEQ ID NO: 17) (SEQ ID

NO: 21

Mma MRSHHHHHHGSMVS LYK 26,063.07 26,059.33 3.74

KG (SEQ ID NO: 18) (SEQ ID

NO: 21 )

Table III: Mass Analysis 2

Calculated monoisotopic masses of the main species of eGFP[Y40am] variants with the proposed N and C-terminal truncations are compared to the experimentally found masses; eGFP chromophore formation was integrated, n.d., not detectable.

eGFP[Y40BocK]

o-pair N_term Cterm rn3SS_ca|_cu|_ated rnaSSf_0unc) Ama ss

Mma MRSHHHHHH LYK (SEQ 26,709.37 26,709.46 0.09

(SEQ ID NO: 14) ID NO: 21 )

Mma MRSHHHHHHG LYK (SEQ 26,652.35 n.d. n.d.

(SEQ ID NO: 19) ID NO: 21 )

Mma MRSHHHHHHGS LYK (SEQ 26,565.32 26,565.40 0.08

(SEQ ID NO: 15) ID NO: 21 )

Mma MRSHHHHHHGSM LYK(SEQ 26,434.28 26434.36 0.08

(SEQ ID NO: 16) ID NO: 21 )

Mma MRSHHHHHHGSMV LYK (SEQ 26,335.21 26,334.34 0.87

(SEQ ID NO: 17) ID NO: 21 )

Mma MRSHHHHHHGSMVSKG LYK (SEQ 26,063.07 26,059.25 3.82

(SEQ ID NO: 20) ID NO: 21 )

Mm MRSHHHHHH LYK (SEQ 26,709.37 26,709.46 0.09

(SEQ ID NO: 14) ID NO: 21 )

Mm MRSHHHHHHG LYK (SEQ 26,652.35 26,652.43 0.08 (SEQ ID NO: 19) ID NO: 21 )

Mm MRSHHHHHHGS LYK (SEQ 26,565.32 n.d. n.d.

(SEQ ID NO: 15) ID NO: 21 )

Mm MRSHHHHHHGSM LYK (SEQ 26,434.28 26434.36 0.08

(SEQ ID NO: 16) ID NO: 21 )

Mm MRSHHHHHHGSMV LYK (SEQ 26,335.21 26334.33 0.88

(SEQ ID NO: 17) ID NO: 21 )

eGFP[Y40AllocK]

Mma MRSHHHHHH LYK (SEQ 26,693.34 26,693.38 0.04

(SEQ ID NO: 14) ID NO: 21 )

Mma MRSHHHHHHG LYK (SEQ 26,636.32 26636.36 0.04

(SEQ ID NO: 19) ID NO: 21 )

Mma MRSHHHHHHGS LYK (SEQ 26,549.29 n.d. n.d.

(SEQ ID NO: 15) ID NO: 21 )

Mma MRSHHHHHHGSM LYK (SEQ 26,418.25 26..418.28 0.03

(SEQ ID NO: 16) ID NO: 21 )

Mma MRSHHHHHHGSMV LYK (SEQ 26,319.18 n.d. n.d.

(SEQ ID NO: 17) ID NO: 21 )

Mm MRSHHHHHH LYK (SEQ 26,693.34 26,693.38 0.04

(SEQ ID NO: 14) ID NO: 21 )

Mm MRSHHHHHHG LYK (SEQ 26,636.32 26,636.33 0.01

(SEQ ID NO: 19) ID NO: 21 )

Mm MRSHHHHHHGS LYK (SEQ 26,549.29 n.d. n.d.

(SEQ ID NO: 15) ID NO: 21 )

Mm MRSHHHHHHGSM LYK (SEQ 26,418.25 26,418.28 0.03

(SEQ ID NO: 16) ID NO: 21 )

Mm MRSHHHHHHGSMV LYK (SEQ 26,319.18 n.d. n.d.

(SEQ ID NO: 17) ID NO: 21 )

eGFP[Y40AzideK]

Mma MRSHHHHHH LYK (SEQ 26,722.34 26,722.42 0.08

(SEQ ID NO: 14) ID NO: 21 )

Mma MRSHHHHHHG LYK (SEQ 26,665.32 26,665.40 0.08

(SEQ ID NO: 19) ID NO: 21 )

Mma MRSHHHHHHGS LYK (SEQ 26,578.29 n.d. n.d. (SEQ ID NO: 15) ID NO: 21 )

Mma MRSHHHHHHGS LYK (SEQ 26,447.25 26,447.33 0.08

(SEQ ID NO: 15) ID NO: 21 )

Mm MRSHHHHHH LYK (SEQ 26,722.34 26,722.37 0.03

(SEQ ID NO: 14) ID NO: 21 )

Mm MRSHHHHHHG LYK (SEQ 26,665.32 26,665.35 0.03

(SEQ ID NO: 19) ID NO: 21 )

Mm MRSHHHHHHGS LYK (SEQ 26,578.29 n.d. n.d.

(SEQ ID NO: 15) ID NO: 21 )

Mm MRSHHHHHHGSM LYK (SEQ 26,447.25 n.d. n.d.

(SEQ ID NO: 16) ID NO: 21 )

Mm MRSHHHHHHGSMV LYK (SEQ 26,348.18 26,348.21 0.03

(SEQ ID NO: 17) ID NO: 21 )

Mm MRSHHHHHHGSMVS LYK (SEQ 26,261 .15 26,263.22 0.07

(SEQ ID NO: 18) ID NO: 21 )

Mm MRSHHHHHH GMDELYK 26,290.20 26,290.24 0.04

(SEQ ID NO: 14) (SEQ ID

NO: 22)

Mm MRSHHHHHHG GMDELYK 26,233.18 26,233.22 0.04

(SEQ ID NO: 19) (SEQ ID

NO: 22)

Mm MRSHHHHHHG LGMDELYK 26, 120.10 26, 120.13 0.03

(SEQ ID NO: 19) (SEQ ID

NO: 23)

In a next step, the MmaPylRS was expressed without the co-expression of tRNA_CUA to assess the orthogonality of the enzyme in E. coli. The enzyme is orthogonal as it did not interfere with the endogenous translation machinery. Particularly, the /W naPyIRS did not charge an E. coli tRNA ( EctRNA) that was able to read through the in-frame TAG codon by wobble base pairing. Independent of the BocK supplementation, a fluorescence signal exceeding the background level (Figure 2) was not detected in the absence of tRNAcu_A- This confirmed that Λ fmaPylRS was orthogonal in E. coli. Also, Pyl is absent from the genetic code of E. coli and naPylRS is neither a member of nor homologous to the E. coli translational machinery. To be orthogonal, MmatRNA_CuA must not be charged with a cAA by an endogenous E. coli aaRS. This was confirmed since in the absence of BocK, a detectable eGFP signal with cells that expressed the MmaPylRS//WmatRNA_CuA pair (Figure 2) was not observed.

It was thus shown that recombinant MmaPylRS and its cognate MmatRNA_CuA suppressed the in-frame TAG codon and introduced BocK into eGFP (Figure 7 and Table II). No cAA was incorporated in response to the amber codon. Thus, the MmaPylRS/MmatRNAcuA is a functional orthogonal pair (o-pair) in E. coli.

For the efficient incorporation of ncAAs (non-canonical amino acids) at amber codons the constitutive expression of the suppressor tRNA_CuA must not interfere with the growth of E. coli. To address this issue, the impact of the constitutive expression of different suppressor tRNAs on the growth of E. coli cells was compared. The growth pattern of cells expressing /WmatRNA_CuA was similar to that of cells expressing either MmtRNAcuA or no tRNAcuA- Also, the expression of MmaOP (/WmaPylRS//WmatRNA_CuA) did not adversely affect the growth of E. coli. The growth was not altered by the supplementation of the cells with different Pyl derivatives BocK, AllocK or AzideK (Figure 6).

The fluorescence readout of amber suppression with BocK using the new MmaPylRS/MmatRNAcuA o-pair (MmaOP; Figure 2) was higher compared to the established MmPylRs/MmtRNA_CUA pair (MmOP).

Example 3: Expression of /lima Pyl RS in E. coli

The expression of Λζ/maPylRS was analyzed in E. coli. The expression of the enzyme from pSCS+ under the arabinose inducible pBAD promoter was only faintly visible in whole cell samples on an SDS gel (not shown). Accordingly, a C-terminal hexahistidine fusion of MmaPyIRS (MmaPylRS-his₆, SEQ ID NO: 7) was designed for facilitated purification and analysis. To assess the impact of the C-terminal his₆-tag on AtfmaPyIRS activity, BocK was incorporated into eGFP[Y40am] (SEQ ID NO: 8) using the o-pairs with /WmaPyIRS or Mma Pyl RS-his₆ and the fluorescence readout was monitored after 4 and 24 h. In accordance with the previous results, a significant fluorescence readout (Figure 3a) was only detectable in the presence of BocK. Cells harboring MmaPylRS- his₆ or /WmaPyIRS showed equal absolute fluorescence after 4 h. Only after 24 hours, the fluorescence of cells carrying MmaPylRS-his₆ was slightly lower compared to the cells with AtfmaPylRS. Hence, it could be confirmed that the C-terminal tagging of the MmaPyIRS has no or only a minor impact on the activity.

Mma Py I RS-his₆ was purified from crude E. coli lysates by Ni²⁺-chelate affinity chromatography. The analysis of the purified protein by SDS-PAGE revealed a band migrating at the expected size of 32 kDa (Figure 3b; lane E, open arrow). The eGFP[Y40am] reporter carried a his₆-tag as well and was co-purified (Figure 3b; lane E, closed arrow). It did not interfere with the analysis of /WmaPylRS-his₆ since both proteins showed distinct migration behavior on the gel. The /WmaPylRS-his₆ band was excised from the gel and peptide mass fingerprinting was performed. /WmaPylRS-his₆ was unequivocally identified by a sequence coverage of approximately 90%. Thus, it could be concluded that the ΛfmaPylRS was expressed in an active form in E. coli. This finding is in agreement with the fluorescence data.

In addition, the eGFP[Y40BocK] was purified from crude lysates of cells expressing the two o-pairs with ΛfmaPylRS and /WmaPylRS-his₆. The cells were supplemented with 5 mM BocK or not. In the presence of BocK, with both o-pairs the successful expression of eGFP[Y40BocK] was visible by a protein band at the calculated molecular weight of 28 kDa (Figure 3b; closed arrow). The insertion of BocK was further confirmed in response to the in-frame amber stop codon of eGFP[Y40am] by mass analysis (Figure 5 and Table II). No cAAs (canonical amino acids) were detected at this position. In contrast, no expression band was visible in the absence of BocK (Figure 3b; green arrow). These results confirm that the MmaOP is functional and orthogonal in E. coli.

Example 4: Differences in ncAA incorporation between AfmaPylRS and AfmPyIRS

The fluorescence readout of cells expressing the MmaOP increased over time in the presence of lysine derivative BocK (Figure 4). Apparently, BocK was better incorporated by the MmaOP than by MmOP (Figure 4). The correct incorporation of BocK into eGFP[Y40am] was confirmed using the MmOP by monoisotopic mass spectroscopy of the purified intact variant proteins. The molecular masses of the incorporated Pyl analog was unambiguously confirmed. Similar to MmOP, the expression of MmaOP did not adversely affect the growth of E. coli. The growth was not altered by the supplementation of the cells with BocK.

The expression of the eGFP variants was confirmed using the different o-pairs by SDS- PAGE. In the presence of BocK, an expression band of eGFP was clearly detectable (not shown). On the contrary, a distinct expression band in the absence of the Pyl analogs was not observed. These results correlated with the fluorescence measurements. In addition, a faint expression band of the MmaPyIRS was detected at 32 kDa in cells induced with arabinose. Most probably, the expressed MmaPyIRS was very active since its weak expression (compared to MmPyIRS) still resulted in effective incorporation of BocK (Figure 4). The expression of the MmPyIRS, however, was much more pronounced compared to MmaPyIRS. The weak expression of a very active o-pair (MmaPyIRS) is advantageous because it uses a minimum of cellular resources for a maximum of activity. To sum up, the results show that the MmaOP appears to be a powerful and improved alternative to the previously described MmOP.

Example 5: Experimental Section

Chemicals and enzymes

All standard chemicals used in this work were purchased from Sigma (St. Louis, MO), Merck KGaA (Darmstadt, Germany) or Carl Roth GmbH (Karlsruhe, Germany), if not stated differently. N_E-Boc-L-lysine (BocK; catalog number: E-1610) was obtained from BACHEM (Bubendorf, Switzerland). Enzymes for cloning and PCR were from Thermo Fisher Scientific (Waltham, MA). PCRs were performed using PhusionOHigh-Fidelity DNA polymerase or Dream Taq®DNA polymerase (Thermo Fisher Scientific) for applications that did not require proof-reading. PCR primers were ordered from IDT Inc. (Coralville, IA) in standard desalted quality. Aqueous stock solutions were sterilized by filtration through 0.20 pm CA syringe filters (Lab Logistic Group GmbH, Meckenheim, Germany). Strains and plasmids

E. coli BL21 (E. coli B F^" ompT gal dcm Ion hsdS_B{rB^~m_B ^~) (ma/S⁺)_K-i2(A^s); Agilent Technologies Inc., Santa Clara, CA) was the host for the stop codon suppression experiments. E. coli BL21 (DE3) (E coli B F^" ompT gal dcm Ion hsdS_B{rB^~mB^~) A(DE3 (lacl lacUV5-T7p07 indl sam7 nin5)) (ma/fi⁺)_K-i2( ^s); Agilent Technologies Inc.) was used for the expression of the AtfmaPylRS-his₆ for crystallography. E. coli Top10F'(E. coli K-12 F{lacP Tn10(tet )) mcrA (mrfhsdRMSmcrBC) cp80/acZAM15 AlacX74 deoR nupG recA^ araD S A(ara-/etv)7697 galil galK rpsL(Str^R) endA^ K; Thermo Fisher Scientific) was used for cloning experiments and plasmid propagation. Transformation of the E. coli strains was carried out by electroporation. The pSCS+ screening plasmid was used as backbone for the cloning of the different PylRS/tRNA_CUA pairs. The coding sequence of the pyrrolysyl-tRNA synthetase from Candidatus Methanomethylophilus alvus (Mma) was PCR-amplified from synthetic DNA (IDT Inc.) using primers BPp1496 (SEQ ID NO: 9) and BPp1497 (SEQ ID NO: 10). The PCR fragment was inserted into pSCS+ plasmid cut with Sail. The different tRNAs_sup from Mma, Methanosarcina mazei {Mm) and Methanocaldoccous janaschii (MJ) were ordered as synthetic DNA (IDT Inc.) flanked by the proK promoter and terminator. The sequences were PCR-amplified with primers BPp81 1 (SEQ ID NO: 1 1 ) and BPp812 (SEQ ID NO: 12) and subsequently cloned into pSCS+ linearized with Pstl. To construct the pET21 a(+)-/WmaPylRS-his₆ plasmid the coding sequence of the MmaPyIRS was PCR-amplified from synthetic DNA (IDT Inc.) using primers BPp1496 (SEQ ID NO: 9) and BPp1498 (SEQ ID NO: 13). The C-terminal hexahistidine tag was introduced by primer BPp1498 (SEQ ID NO: 13). The PCR fragment was inserted into pET21 a(+) (Qiagen) cut with Xbal and Hindlll. All inserts were cloned into the linearized vectors by Gibson isothermal assembly⁴³). All constructs were sequence verified (Microsynth, Vienna, Austria).

Cultivation conditions Stop codon suppression (SCS)

Lysogen broth (LB) medium (Roth) was used for all stop codon suppression experiments. E. coli BL21 cells harboring a pSCS+ plasmid carrying an o-pair consisting of a PylRS and a tRNA_CuA were cultivated in 250 mL flasks each containing 50 mL LB medium with 50 μg/mL kanamycin (Roth). The initial cell density D₆₀₀ was 0.1. Cultures were incubated at 37 °C on an orbital shaker at 160 rpm. At D₆₀₀ of 0.8-1.0, the expression of the PylRS was induced by adding 0.2% (w/v) of arabinose (Roth). In addition, either 5 mM of the corresponding Pyl analog dissolved in 10 mM sodium phosphate buffer pH 8.0 pair, or the same volume of buffer (fidelity controls, without Pyl analog) were supplemented. Cells corresponding to 0.5 D₆₀o values were harvested and dissolved in 4 X SDS sample buffer (200 mM Tris-HCI pH 6.8, 400 mM DTT, 8% (w/v) SDS, 0.4% (w/v) bromphenol blue, 40% (w/v) glycerol) for subsequent SDS-PAGE.

Expression of the MmaPylRS-his6 for crystallography

E. coli BL21 (DE3) cells harboring the pET21 a(+)-MmaPylRS-h₆ plasmid were cultivated in 1000 mL flasks each containing 400 mL LB medium with 50 g/mL kanamycin (Roth). The initial cell density D₆₀₀ was 0.1. Cultures were incubated at 37 °C on an orbital shaker at 160 rpm. At D₆₀₀ of 0.8-1.0, the expression of the /WmaPylRS-h₆ was induced by adding 0.5 mM of IPTG (Biosynth, Staad, Switzerland). MmaPylRS expression was conducted at 30 °C on an orbital shaker at 160 rpm for 20 hours. Cells were harvested at 5,000 g for 20 minutes at 4 °C. Analytics

Cell growth was monitored by reading the attenuance at 600 nm (D₆oo) using an Eppendorf BioPhotometer® (Eppendorf, Wesseling-Berzdorf, Germany). For SCS experiments, eGFP fluorescence and (A_ex=488 nm; A_em=508 nm) and D₆₀₀ measurements were performed on a synergyMx SMATBLD(+) Gen5 SW plate reader (SZABO-SCANDIC, Vienna, Austria). Cells were diluted 1 :5 (v/v) with 1 x PBS buffer (137 mM NaCI, 2.7 mM KCI, 10 mM Na₂HP0₄, 2 mM KH₂P0₄) and 8 times 100 μΙ_ were measured in a NUNC flat bottom 96-well black plate (Thermo Fisher Scinetific) after 5 seconds of vigorous shaking. SDS-PAGE using 4-12% Bis-Tris SDS-gels (Thermo Fisher Scientific) was performed following the instructions of the manufacturer.

Protein purification The cells were mechanically lysed on a Homogenizer Emulsiflex C3 (Avestin, Ottawa, Canada) and the cell lysates were subsequently centrifuged at 40,000 x g at 4 °C for 60 minutes. The his-tagged proteins were further purified via Ni²⁺-chelate affinity chromatography using Ni-NTA agarose following the instructions of the manufacturer (Qiagen, Hilden, Germany). Samples were desalted using a Sephadex G-25 in PD-10 Desalting Column (GE Healthcare, Chicago, IL) or HiPrep 26/10 Desalting (GE Healthcare).

Click reaction Purified eGFP (roughly 0.5-1 mg/mL) protein solutions were incubated with 0.5 mM copper sulfate, 5 mM sodium ascorbate, 2.5 mM THPTA (tris(3-hydroxypropyltriazolyl- methyl)amine; catalog number: 762342, Sigma Aldrich) and 20 μΜ TAMRA-alkyne (5- carboxytetramethylrhodamine, A_ex 556 nm; A_em 563 nm; catalog number: CLK-059, Jena Bioscience, Jena, Germany). The reaction mixture was incubated over night at 4 °C as described in Uttamapinat et al., Angew Chem Int Ed (2012), 51 (24): 5852-5856. Samples were heated to 98 °C for 10 minutes prior SDS-PAGE analysis. TAMRA labeled eGFP samples were separated on a 4-12% Bis-Tris gel following the instructions of the manufacturer (Thermo Fisher Scientific). The gels were exposed to UV-light (λ 312-365 nm) to detect TAMRA fluorescence and subsequently stained with Coomassie Blue following standard procedures.

Mass analysis

Peptide sequencing by tandem mass spectrometry

The excised protein bands from the SDS gel were reduced, alkylated and digested with Promega modified trypsin according to the method of Shevchenko et a/..(Shevchenko, Analytical Chemistry (1996), 68(5): 850-858). Peptide extracts were dissolved in 0.1 % (v/v) formic acid, 5% (v/v) acetonitrile and separated by nano-HPLC (Dionex Ultimate 3000) equipped with a C₁₈, 5 prri, 100 A, 5 x 0.3 mm enrichment column and an Acclaim PepMap RSLC nanocolumn (C₁₈, 2 pm, 100 A, 500 x 0.075 mm) (all Thermo Fisher Scientific). Samples were concentrated on the enrichment column for 2 min at a flow rate of 5 μΙ/min with 0.1 % (v/v) heptafluorobutyric acid as isocratic solvent. Separation was carried out on the nanocolumn at a flow rate of 300 nl/min at 60 °C using the following gradient, where solvent A is 0.1 % (v/v) formic acid in water and solvent B is acetonitrile containing 0.1 % (v/v) formic acid: 0-2 min: 4% B; 2-90 min: 4-25% B; 90-95 min: 25-95% B, 96-1 10 min: 95% B; 1 10-1 10.1 min: 4% B; 1 10.1-125 min: 4% B. The Bruker maXis II ETD mass spectrometer was operated with the captive source in positive mode with following settings: mass range: 200-2000 m/z, 2 Hz, precursor acquisition control top17, capillary 1300 V, dry gas flow 3 L/min with 150 °C, nanoBooster 0.2 bar. The LC-MS/MS data were analyzed by the Bruker Data analysis software using the Sum Peak algorithm, and searched against a database containing the protein sequence of interest and all common contaminants with Bruker Proteinscape 4.0. Carbamidomethylation on Cys was entered as fixed modification, oxidation on methionine as variable modification. Detailed search criteria were used as follows: trypsin, max. missed cleavage sites: 2; search mode: MS/MS ion search with decoy database search included; precursor mass tolerance +/- 10 ppm; product mass tolerance +/- 0.02 Da; acceptance parameters for identification: 1 % protein FDR and min. 2 peptides per protein.

Intact protein mass analysis

The protein solutions were desalted using Amicon Ultra 0.5 mL Centrifugal filter units (Millipore, Billerica, MA). A final protein concentration of 10 ng/pL in 5% (v/v) acetonitrile and 0.1 % (v/v) formic acid (in water) was obtained. Protein species were separated by nano-HPLC (Dionex Ultimate 3000) equipped with a Pepswift precolumn (monolithic, 5 x 0.2 mm) and a ProSwift RP-4H column (monolithic, 100 μιη χ 25 ατι) (all Thermo Fisher Scientific). 1 μΙ of protein sample was injected and concentrated on the enrichment column for 2 min at a flow rate of 5 μΙ/min with 0.1 % (v/v) heptafluorobutyric acid as isocratic solvent. Separation was carried out on the nanocolumn at a flow rate of 1 pL/min at 37 °C using the following gradient, where solvent A is 0.1 % (v/v) formic acid in water and solvent B acetonitrile containing 0.1 % (v/v) formic acid: 0-2 min: 5% B; 2- 17 min: 5-60% B; 17-20 min: 60% B; 20-20,1 min: 60-5% B; 20,1-29 min: 5% B. The maXis II ETD mass spectrometer (Bruker, Bremen, Germany) was operated with the captive spray source in positive mode with following settings: mass range: 300- 3000 m/z, 1 Hz, source voltage 1.6 kV, dry gas flow 3 L/min at 180 °C. The protein mass spectra were deconvoluted by Bruker Data analysis software, using the MaxEnt2 algorithm. The following main parameters were applied: charge carrier, H+, m/z range, min. 800 to max. 2000, min. instrument resolving power was set to 50,000. For peak detection SNAP algorithm with following parameters were used: Quality factor threshold 0.9, S/N threshold 2 and maximum charge state of 12.

Claims

1. Polynucleotide comprising a nucleotide sequence which is at least 70% identical to the nucleotide sequence according to SEQ ID NO: 1 ,

wherein said polynucleotide encodes an amino acyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic or aliphatic amino acid to form an aminoacyl-tRNA,

wherein said nucleotide sequence being at least 70% identical to the nucleotide sequence according to SEQ ID NO: 1 is codon-optimized to a selected host cell, wherein said host cell is not Methanomethylophilus alvus Mx1201 Ca.

2. Polynucleotide of claim 1 , wherein said nucleotide sequence being at least 70% identical to the nucleotide sequence according to SEQ ID NO: 1 comprises at least 1 of the following substitutions or pair of substitutions a) to q):

a) C39G

b) C87G

c) A94C and G96T

d) A343C and G345T

e) C351 G

f) A352C and G354T

g) A388C and G390T

h) A397C and G399T

i) A448C and G450T

j) C501 G

k) C579G

I) C594G

m) C687G

n) C738G

o) A757C and G759T

p) C807G, and/or

q) G827A.

3. Polynucleotide of claim 1 or 2, wherein said nucleotide sequence being at least 70% identical to the nucleotide sequence according to SEQ ID NO: 1 comprises only nucleotide deviations compared to nucleotides 1 to 825 of SEQ ID NO: 1 which result in one or more silent mutations, or conservative or highly conservative amino acid substitutions when being expressed to a polypeptide.

4. Polynucleotide of any of the preceding claims, wherein said amino acid is a lysine derivative.

5. Polynucleotide of claim 4, wherein said lysine derivative is selected from the group consisting of pyrrolysine, boc-lysine, alloc-lysine, azide-lysine, 2-N.6-N- Bis(2,3-dihydroxy-N-benzoyl)-L-serine, 2-N,6-N-Bis(2,3-dihydroxy-N-benzoyl)-L- serine amide, 3-hydroxylysine, /\/-benzoylglycyl-/\/⁶-[2-hydroxy-2-(3- methylquinoxalin-2-yl)ethyl]lysine, /V-benzoylglycyl-A^-^-hydroxy-S-iquinoxalin^- yl)propyl]lysine, /V-hippuryl-A^-icarboxymethylJIysine, A^-^^-dinitrophenylJIysine, A/⁶-(2-carboxyethyl)lysine, /N^-acetonyllysine, A^-carbamoylmethyllysine, Λ/⁶- methyllysine, hydroxylysine, isodesmosine, ornithine derivatives such as 2- amino-5-(prop-2-ynoylamino)pentanoic acid (5-(prop-2-ynoylamino)ornithine), and 2-amino-5-[(azidoacetyl)amino]pentanoic acid.

6. Polynucleotide of any of the preceding claims, which comprises or consists of a nucleotide sequence according to SEQ ID NO: 3.

7. Vector comprising a polynucleotide comprising a nucleotide sequence which is at least 70% identical to the nucleotide sequence according to SEQ ID NO: 1 , wherein said polynucleotide encodes an amino acyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic or aliphatic amino acid to form an aminoacyl-tRNA.

8. Host cell comprising a vector of claim 7 or a polynucleotide comprising a nucleotide sequence which is at least 70% identical to the nucleotide sequence according to SEQ ID NO: 1 ,

wherein said host cell is not Methanomethylophilus alvus Mx1201 Ca.

9. Host cell of claim 8, wherein said polynucleotide is a polynucleotide according to any one claims 1 to 6.

10. Host cell of claim 8 or 9, which further comprises a polynucleotide encoding a tRNA in addition to said aminoacyl tRNA synthetase encoded by said polynucleotide comprised by the host cell, said tRNA and aminoacyl tRNA synthetase being an orthogonal pair.

1 1. Polynucleotide of any one of claims 1 to 6 or host cell of any one of claims 8 to 10, wherein said host cell does not belong to the domain of archaea.

12. Polynucleotide of any one of claims 1 to 6, or host cell of any one claims 8 to 1 1 , wherein said host cell is a prokaryote or eukaryote.

13. Polynucleotide or host cell of claim 12, wherein said host cell is selected from the group consisting of alia E. coli, Mycoplasma capricolum, CHO, SF9 cells, C. elegans cell, S. cerevisiae, Schizosaccharmyces pombe, Micrococcus luteus, Komagataella pastoris (aka Komagataella phaffii), and Bombyx mori.

14. Polypeptide encoded by the polynucleotide of any one of claims 1 to 6, or 1 1 to 13.

15. Composition comprising aminoacyl tRNA synthetase as defined in claim 14 and tRNA as orthogonal pair.

16. Use of a polynucleotide of any one of claims 1 to 6, 1 1 to 13, a vector of claim 7, a polypeptide of claim 14, or a composition of claim 15 to catalyze the introduction of an aromatic or aliphatic amino acid into a polypeptide.

17. Method of incorporation of an aromatic or aliphatic amino acid into a polypeptide, comprising the following steps:

(i) expressing a polynucleotide comprising a nucleotide sequence which is at least 70% identical to the nucleotide sequence according to SEQ ID NO: 1 , wherein said polynucleotide encodes an amino acyl tRNA synthetase capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic or aliphatic amino acid to form an aminoacyl-tRNA in a host cell, wherein said host cell is not Methanomethylophilus alvus Mx1201 Ca, and expressing a polynucleotide encoding a tRNA, said tRNA and aminoacyl tRNA synthetase being an orthogonal pair.