Synthesis of 1n-methylpseudouridine and 1n-methylpseudouridine phosphates

Abstract

A method of synthesising a 1N-alkyl derivative of pseudouridine comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine. The substrate has a structure of: Formula (I). The 1N-methyl derivative of pseudouridine has a structure of: Formula (II). R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group. R2 is H or a hydroxyl group. R3 is a methyl group or an ethyl group. The cofactor is a source of a methyl group or an ethyl group. The methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate. Enzymatic synthesis of 1N-methyl derivatives of pseudouridine may be more efficient and produce less waste than chemical syntheses. Also provided are methyltransferases and buffers useful in the method; and polynucleotides, expression vectors, and microorganisms useful for producing the methyltransferases.

Description

[0001]Synthesis of 1N-methylpseudouridine and 1N-methylpseudouridine phosphates Background 5 1N-methylpseudouridine (1N-methyl-Ψ) nucleoside and its various phosphorylated derivatives (such as 1N-methylpseudouridine-5’-monophosphate (1N-methyl-ΨMP) and 1N- methylpseudouridine-5’-triphosphate (1N-methyl-ΨTP)) are used instead of uridine in mRNA- based vaccines in order to reduce immune response to the mRNA molecules and improve overall vaccine efficiency. For example, N1-methylpseudouridine is used instead of uridine in 10 Pfizer-BioNTech’s mRNA vaccine against COVID-19, BNT162b2. The rapid adoption of mRNA-based vaccines and expansion of their application into different clinical areas creates high commercial demand for 1N-methylpseudouridine nucleoside and its derivatives. 15 One existing process for synthesising 1N-methylpseudouridine and its derivatives is illustrated in Fig.1. Pseudouridine 101 is obtained from natural sources. Chemical methylation is performed to obtain 1N-methylpseudouridine 102. Chemical methylation involves protection and deprotection steps in addition to the use of a methylating agent such as methyl iodide. 20 1N-methylpseudouridine monophosphate 103 and 1N-methylpseudouridine triphosphate 104 are obtainable by chemical phosphorylation of the 1N-methylpseudouridine 102. An alternative existing process involves extracting 1N-methylpseudouridine from natural sources. 25 Both existing processes require multiple steps to complete, and produce unwanted waste. As the mRNA vaccine market grows, there is an increasing demand for cleaner, cheaper, and quicker ways to produce the modified nucleosides and their derivatives. Summary In one aspect, there is provided a method of synthesising a 1N-alkyl derivative of pseudouridine. The method comprises contacting a substrate with a methyltransferase in the 5 presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine. The substrate has a structure of: , and the 1N-methyl derivative of pseudouridine has a structure of: 10 . In the formulae above, R1is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group; R2is H or a hydroxyl group; and R3is a methyl group or an ethyl group. 15 The cofactor is a source of a methyl group or an ethyl group. The methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate. Further aspects provide methyltransferases useful in the method; polynucleotides encoding the methyltransferases; expression vectors carrying the polynucleotides; and microorganisms transduced with the expression vectors. 5 Still another aspect provides the use of an aqueous buffer as a reaction medium for the synthesis of the 1N-alkyl derivative of pseudouridine in accordance with the method defined above. The aqueous buffer includes Mg2+at a concentration in the range 0.5 mM to 15 mM, and has a pH in the range 7.4 to 7.6. 10 This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Nor is the claimed subject matter limited to implementations that solve any or all of the disadvantages noted herein. 15 Brief Description of the Drawings To assist understanding of embodiments of the present disclosure and to show how such embodiments may be put into effect, reference is made, by way of example only, to the 20 accompanying drawings in which: Fig. 1 is a reaction scheme outlining a comparative process for synthesising 1N- methylpseudouridine and phosphorylated derivatives thereof. Fig.2 is a bar chart showing the concentration of S-adenosylhomocysteine “SAH” 25 detected when the assay described in Example 1 was performed using enzyme N1 in various different buffer systems. Fig. 3 is a bar chart showing the concentration of SAH detected when the assay described in Example 1 was performed using enzyme N1 in the presence of (a) Ψ, (b) ΨMP, (c) ΨTP, and (d) no substrate. 30 Figs 4A to 4C are mass chromatograms of reaction mixtures containing enzyme N1 and various different substrates, following treatment with an alkaline phosphatase, as described in Example 1. Fig. 4A is a mass chromatogram obtained when pseudouridine was used as the substrate. Fig.4B is a mass chromatogram obtained when pseudouridine-5’-monophosphate was used as the substrate. Fig.4C is a mass chromatogram obtained when pseudouridine-5’-triphosphate was used as the substrate. 5 Fig.5 (a comparative example) shows a mass chromatogram of commercially-available 1N-methylpseudouridine standard. Figs 6A to 6C are a series of mass chromatograms that confirm that the present method produced 1N-methylpseudouridine. The chromatograms are of a reaction mixture containing enzyme N1 and pseudouridine-5’-monophosphate. Fig.6A is a 10 mass chromatogram of the reaction mixture alone. Fig.6B is a mass chromatogram of the reaction mixture spiked with 5 picomoles of 1N-methylpseudouridine standard. Fig.6C is an overlayed view of the unspiked reaction mixture and the same reaction mixture spiked with 5 picomoles of 1N-methylpseudouridine standard. Fig. 7 is a pairwise identity matrix of natural (N) and engineered (E) SPOUT 15 methyltransferases. Each of these enzymes was found to be effective for methylating pseudouridine and its derivatives. Fig.8 is a bar chart showing concentrations of SAH detected when methylation of 2'- deoxypseudouridine was performed using enzymes N1, E5 and E8, as described in Example 3. 20 Fig.9A shows the concentration of SAH detected when ethylation of Ψ and ΨMP was conducted using enzyme E5, as discussed in Example 4. Fig. 9B shows the concentration of SAH detected when ethylation of ΨMP was conducted using enzyme E6, as discussed in Example 4. Fig.10 is a pairwise identity matrix for four mRNA cap guanine-N7 methyltransferases 25 S1 to S4. Fig.11 is a reaction scheme outlining an example method of synthesising 1N-methyl psuedouridine from uridine. Fig.12 shows photographs of gels used to purify the proteins discussed in Example 7. M is a protein molecular weight ladder, and arrows indicate purified proteins. 30 Fig.13 is a bar chart showing concentrations of SAH detected when a pseudouridine substrate was methylated using the proteins discussed in Example 7. Fig.14 is a set of mass chromatograms for a reaction mixture comprising enzyme S17, as discussed in example 7. Fig.15 is a set of mass chromatograms for a control mixture, as discussed in Example 7. 5 Detailed Description General definitions 10 The verb ‘to comprise’ is used herein as shorthand for ‘to include or to consist of’. In other words, although the verb ‘to comprise’ is intended to be an open term, the replacement of this term with the closed term ‘to consist of’ is explicitly contemplated, particularly where used in connection with chemical compositions. 15 The symbol “Ψ” refers to pseudouridine. “ΨMP” refers to pseudouridine monophosphate. “ΨTP” refers to pseudouridine triphosphate. ΨTP “dΨ” refers to deoxypseudouridine. “dΨMP” refers to deoxypseudouridine monophosphate. “dΨTP” refers to deoxypseudouridine triphosphate. dΨTP 5 “SAM” stands for S-adenosyl-L-methionine: SAM “SAH” stands for S-adenosylhomocysteine. As used herein, the expression “1N-alkyl derivative of pseudouridine” refers to a compound of Formula 2: Formula 2 where: R1is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group; R2is H or a hydroxyl group; and R3is a methyl group or an ethyl group. The methods provided herein most typically produce 1N-methyl derivatives of pseudouridine, i.e. compounds of Formula 2 in which R3is a methyl group. "Phosphorylated” compounds are compounds in which R1is a monophosphate group, a diphosphate group, or a triphosphate group. Monophosphates and triphosphates are typically preferred. As used herein, the term “phosphate” encompasses monophosphates and polyphosphates unless context clearly dictates otherwise. Mutations are described using the nomenclature of den Dunnen and Antonarakis: Hum Genet 109(1): 121-124 (also published online as https: / / web.archive.org / web / 20221111075857 / https: / / www.hgmd.cf.ac.uk / docs / mut_nom .html). For example, taking SEQ ID NO: 1 as a parent sequence, “K10Q” means replace the K residue at position 10 with a Q residue; "C203del” means delete residue C203; and E149_N150insN means insert an N residue between residues E149 and N150. Position numbering refers to the parent sequence. Insertions and deletions do not shift the numbering of subsequent residues. 5 The word “about” when used in connection with a numerical value encompasses values within ± 10 % of the stated value. As used herein, “positively charged residues” refers to histidine (H), lysine (K), and arginine (R) residues. 10 Where it is said that a sequence includes mutations, the mutations may optionally be limited to those which are specifically identified herein. Methods of synthesising 1N-alkyl derivatives of pseudouridine 15 Provided herein is a method of synthesising a 1N-alkyl derivative of pseudouridine. The method comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine. 20 The method may be performed in vivo or ex vivo. The substrate has a structure of Formula 1: Formula 1 R1is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group, and R2is H or OH. In other words, the substrate is an optionally-phosphorylated pseudouridine or deoxypseudouridine. 5 Atom O4 of the substrate is bolded in the formula above. This atom may be involved in the binding of the substrate to the enzyme. More specifically, atom O4 may interact with a positively-charged residue in the active site of the methyltransferase. The source of the substrate is not particularly limited. The substrate may be extracted from 10 natural sources, formed in vivo, or chemically synthesised. Chemical phosphorylation of pseudouridine or deoxypseudouridine may be performed. The substrate is monomeric, and is not part of an RNA, DNA, or the like. 15 The substrate may be produced by: i) contacting uridine with a uridine phosphorylase in the presence of a monophosphate ion source to obtain uracil and α-D-ribose-1-monophosphate; ii) contacting the α-D-ribose-1-phosphate with a pentomutase to obtain D-ribose-5- monophosphate; 20 iii) contacting the D-ribose-5-monophosphate with pseudouridine monophosphate glycosidase to form pseudouridine 5’-monophosphate; and optionally iv) contacting the pseudouridine 5’-monophosphate with a phosphatase to obtain pseudouridine. 25 Steps i) to iii) and optionally iv) may be performed in vivo, particularly in implementations where the synthesis of the 1N-alkyl derivative of pseudouridine is performed in vivo. The 1N-alkyl derivative of pseudouridine produced by the method has a structure of Formula 2: Formula 2 5 Contacting the substrate with the methyltransferase does not modify R1or R2. R3is a methyl group or an ethyl group, depending upon the cofactor chosen. The nature of the cofactor is not particularly limited, provided that the cofactor acts as a source of the methyl group or ethyl group, and the methyltransferase is capable of 10 transferring the methyl group or ethyl group from the cofactor to the substrate to produce the 1N-alkyl derivative of pseudouridine. The methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate. Numerous examples of suitable methyltransferases are provided 15 hereinbelow. As will be demonstrated in the Examples, the primary structures of methyltransferases vary widely. By way of illustration, Fig. 6 shows that, even with a specific family of methyltransferases (in this example, SPOUT methyltransferases) methyltransferases which 20 are effective for transferring methyl groups to substrates of Formula 1 can share less than 20 % sequence identity. Methyltransferases which methylate pseudouridine bases of RNA may be suitable, and can be identified by searching publicly-available databases. The activity of such 25 methyltransferases may be improved by introducing one or more mutations, e.g. to allow improved binding of the substrate via a stacking interaction and / or to provide an active site having neutral or positive charge to stabilise the deprotonated form of the substrate. Methyltransferases useful in the present method will typically have one, and preferably both, of the following properties: i) an active site which includes a positively-charged residue positioned to interact with atom O4 of the pseudouridine base, the positively-charged residue being selected from lysine and arginine; and / or ii) a stacking amino acid residue selected from a histidine residue, a tyrosine residue, and a tryptophan residue, arranged proximal to the active site and capable of forming a stacking interaction with the pseudouridine base. In implementations where R1is a monophosphate group, a diphosphate group, or a triphosphate group, the methyltransferase may also usefully include: iii) a salt bridging amino acid residue selected from lysine and arginine, the salt bridging amino acid residue being arranged proximal to the active site and capable of forming a salt bridge with the monophosphate group, diphosphate group, or triphosphate group. Optionally, the cofactor is S-adenosyl-L-methionine or S-adenosyl-L-ethionine. Most typically, the methods provided herein produce a 1N-methyl derivative of pseudouridine (i.e., R3is a methyl group). The cofactor is most typically S-adenosyl-L- methionine. Optionally, R1is selected from a hydroxyl group, a monophosphate group, and a triphosphate group. When R1is a hydroxyl group or a monophosphate group, the method may further comprise, after the contacting, phosphorylating the 1N-alkyl derivative of pseudouridine. The phosphorylation may be chemical phosphorylation. R2may be a hydroxyl group. In other words, the substrate may be an optionally- phosphorylated pseudouridine. Alternatively, R2may be H. In other words, the substrate may be an optionally- phosphorylated deoxypseudouridine. The method may further comprise producing the methyltransferase by fermentation of a microorganism configured to express the methyltransferase. The contacting may be performed in the presence of the microorganism. In such implementations, the substrate may be contacted with the methyltransferase inside or outside the microorganism. The synthesis of the 1N-alkyl derivative of pseudouridine may take place in vivo. In such implementations, the 1N-alkyl derivative of pseudouridine may be purified from cell media or biomass. Performing the synthesis in vivo may avoid a need to extract and purify the methyltransferase, thereby improving efficiency. The contacting may be performed in an aqueous buffer, such as a Tris buffer. The aqueous buffer may include Mg2+at a concentration in the range 0.5 mM to 15 mM, optionally 0.7 mM to 1.3 mM, further optionally 0.9 to 1.1 mM. The Mg2+may be provided by any suitable magnesium salt, such as MgCl2. Experiments reported in Example 1, below, demonstrated that including Mg2+in the buffer increased the activity of the methyltransferase. The buffer may have a pH in the range 7.4 to 7.6. Methyltransferases used in the Examples were found to be most active at approximately pH 7.5, though other pHs can be used. The buffer may further include: NaCl at a concentration of 50 to 350 mM, optionally 270 to 230 mM, further optionally 290 to 310 mM; and / or KCl at a concentration of 70 to 130 mM, optionally 90 to 110 mM. The concentration of the substrate, cofactor, and methyltransferase in the buffer are not particularly limited and may be selected as appropriate. For example, the cofactor may be 5 present at a concentration in the range 20 to 30 µM, and the substrate may be present at a concentration in the range 80 to 120 µM. The concentration of the methyltransferase is typically in the range 0.5 to 20 µM. In particular, the buffer may have a pH of 7.5 ± 0.1 and include: 10 Tris-HCl at a concentration of about 25 mM; NaCl at a concentration of about 300 mM; MgCl2 at a concentration of about 1 mM; KCl at a concentration of about 100 mM; the cofactor (e.g., SAM) at a concentration of about 25 µM; and 15 the substrate at a concentration of about 100 µM. One aspect provides a use of the buffers discussed above as a reaction medium for the enzymatic synthesis by a methyltransferase of a 1N-alkyl derivative of pseudouridine according to Formula 2 from a substrate of Formula 1 and a cofactor selected from S- 20 adenosyl-L-methionine and S-adenosyl-L-ethionine. The transfer of the methyl group or ethyl group from the cofactor to the substrate forms a dealkylated cofactor, e.g. S-adenosylhomocysteine when the cofactor is S-adenosyl-L- methionine or S-adenosyl-L-ethionine. The method may optionally include regenerating the 25 cofactor from its dealkylated derivative. Regenerating the cofactor may improve the yield and / or rate of reaction. Regenerating the cofactor in effect increases the amount of cofactor available. SAM is expensive and unstable, and regenerating SAM may therefore improve the cost-efficiency of the method. Additionally, without wishing to be bound by theory, SAH may act as an inhibitor of methyltransferases and removing SAH may therefore be desirable. 30 The way in which the cofactor is regenerated is not particularly limited and may be selected as appropriate. The cofactor may be regenerated using an alkyl group donor in the presence of a catalyst, typically an enzyme. 5 The alkyl group donor may be an alkyl halide and the catalyst may be a methyl halide transferase. For example, the cofactor may be S-adenosyl-L-methionine, the alkyl halide may be methyl iodide, and the methyl halide transferase may be a methyl halide transferase from Chloracidobacterium thermophilum. The sequence of this protein is recorded in UniProt (entry name: G2LF24_CHLTF; database: TrEMBL; release no.2024_02 / 2024_02, release date 10 27 March 2024). The sequence of this protein is: MLGMDADTASFWEEKYRADLTAWDRGGVSPALEHWLAEGALKPGRILIPGCGYGHEVLALAR RGFEVWGLDIALTPVRRLQEKLAQAGLTAHVVEGDVRTWQPEQPFDAVYEQTCLCALSPEDW PRYEAQLCRWLRPGGRLFALWMQTDRPGGPPYHCGLEAMRVLFALERWRWVEPPQRTVPH PTGFFEYAAILERLV (SEQ ID NO: 29). 15 In accordance with another possibility, the alkyl group donor may be an alkyl toluenesulfonate and the catalyst may be a thiopurine alkyltransferase. For instance, the alkyl group donor may be methyl p-toluenesulfonate and the thiopurine alkyltransferase may be a thiopurine methyltransferase from Ustilago maydis. The sequence of this protein is recorded in UniProt 20 (entry name: A0A0D1DT00_USTMA; database: TrEMBL, release no. 2024_02 / 2024_02, release date 27-Mar-2024) and is: MTSSLSKDDQIQNLRRLFADSGVPNDPKAWDQAWIDSTTPWDANRPQPALVELLEGAHDAD AKVPDVDGNLIPVSQAIPKGDGTAVVPGCGRGYDARVFAERGLTSYGVDISSNAVAAANKWLG DQDLPTELDDKVNFAEADFFTLGTSKSLVLELSKPGQATLAYDYTFLCAIPPSLRTTWAETYTRLL 25 AKHGVLIALVFPIHGDRPGGPPFSISPQLVRELLGSQKNADGSAAWTELVELKPKGPETRPDVER MMVWRRS (SEQ ID NO: 30). 30 The methyltransferase used in the method may be a Class I methyltransferase engineered to transfer the methyl group or ethyl group from the cofactor to the substrate. For example, the methyltransferase may be a cap methyltransferase, in particular an mRNA cap guanine- N7 methyltransferase. The methyltransferase may have an active site with a neutral charge or a positive charge. In 5 particular, the methyltransferase may be an engineered mRNA cap guanine-N7 methyltransferase which includes one or more mutations to provide an active site which is more positively-charged than the active site of the wild-type mRNA cap guanine-N7 methyltransferase. 10 For example, the mutations may include replacing a negatively-charged glutamic acid or aspartic acid residue in the active site (most typically, a glutamic acid residue) with a neutrally- charged residue selected from asparagine (N), serine (S), cysteine (C), alanine (A), threonine (T), methionine (M), glycine (G), valine (V), leucine (L), or isoleucine (I). 15 Alternatively, the mutations may include replacing one or more negatively-charged glutamic acid or aspartic acid residue in the active site (most typically, a glutamic acid residue) with a positively-charged residue selected from arginine (R), lysine (K), and histidine (H). Of these, arginine is preferred. 20 The mutations may alternatively or additionally include replacing one or more neutrally- charged residue (such as an F, Y, or I residue) with a positively-charged residue selected from arginine, lysine and histidine; optionally arginine or lysine; and preferably arginine. Providing positive charge in the active site has been found to enhance activity. Particularly 25 preferably, the positive charge is positioned to interact with atom O4 of the substrate. Excessive positive charge may however reduce the stability of the enzyme. It is therefore preferable for the active site to have a net positive charge of one or two. Further positively- charged residues may optionally be present away from the active site, for instance to provide a salt bridge to the substrate, provided that acceptable stability is maintained. 30 Examples of engineered mRNA cap guanine-N7 methyltransferases modified to have positively charged active sites are methyltransferases of SEQ ID NO: 5 having one or more mutations selected from: i) E212Q, E212K, E212R, E212N, E212S, E212C, E212A, E212T, E212M, E212H, E212G, 5 E212V, E212L, and E212I; optionally E212K or E212R, and preferably E212R; ii) F201R or F201K; iii) I267R or I267K; iv) Y271R or Y271K. 10 Typically, the mutations introduce at least one but no more than two positively charged residues. Any one of the mutations at position 212 that do not introduce a positive charge (E212Q, E212N, E212S, E212C, E212A, E212T, E212M, E212G, E212V, E212L, and E212I) may be combined with one or two mutations selected from groups ii) to iv). 15 Further examples of engineered mRNA cap guanine-N7 methyltransferases modified to have positively charged active sites are methyltransferases of SEQ ID NO: 6 having one or more mutations selected from: i) E226Q, E226K, E226R, E226N, E226S, E226C, E226A, E226T, E226M, E226H, E226G, E226V, E226L, or E226I; optionally E226K or E226R; and preferably E226R; 20 ii) F215R or F215K; iii) A286R or A286K; iv) Y290R or Y290K. Typically, the mutations introduce at least one but no more than two positively-charged 25 residues. Any of the mutations at position 226 that do not introduce a positive charge may be combined with one or two mutations selected from groups ii), iii), to iv). Still further examples of engineered mRNA cap guanine-N7 methyltransferases modified to have positively charged active sites are methyltransferases of SEQ ID NO: 7 having one or 30 more mutations selected from: i) E235Q, E235K, E235R, E235N, E235S, E235C, E235A, E235T, E235M, E235H, E235G, E235V, E235L, or E235I; optionally E235K or E235R; and preferably E226R; ii) Y224R or Y224K; iii) A299R or A299K; iv) Y304R or Y304K. 5 Typically, the mutations introduce at least one but no more than two positively-charged residues. Any of the mutations at position 235 that do not introduce a positive charge may be combined with one or two mutations selected from groups ii), iii), to iv). Additional examples of engineered mRNA cap guanine-N7 methyltransferases modified to 10 have positively charged active sites are methyltransferases of SEQ ID NO: 8 having one or more mutations selected from: i) E210Q, E210K, E210R, E210N, E210S, E210C, E210A, E210T, E210M, E210H, E210G, E210V, E210L or E210I; optionally E210K or E210R; and preferably E210R; ii) F199R or F199K; 15 iii) V265R or V265K; iv) Y269R or Y269K. Typically, the mutations introduce at least one but no more than two positively-charged residues. Any of the mutations at position 210 that do not introduce a positive charge may 20 be combined with one or two mutations selected from groups ii), iii), to iv). Also contemplated are variants of the various engineered mRNA cap guanine-N7 methyltransferases described above, the variants having at least 80 % total sequence identity to the corresponding wild-type sequence (SEQ ID NO: 5, 6, 7, or 8). 25 The Class 1 methyltransferase may alternatively be engineered from a xanthosine methyltransferase. Methyltransferases engineered from xanthosine methyltransferases may have high activity. 30 The methyltransferase may have an amino acid sequence which includes a portion having at least 90 % sequence identity to residues 1 to 306 of SEQ ID NO: 19. This amino acid sequence was highly conserved in two representative xanthosine methyltransferases from Coffea spp., S5 and S6 as discussed in the Examples below. The methyltransferase may have an amino acid sequence with at least 90 % sequence identity 5 to SEQ ID NO: 19, the amino acid sequence including one or more mutations selected to increase stability and / or activity compared with the methyltransferase of SEQ ID NO: 19. The mutations selected to increase activity may comprise Q161H and / or A238S. 10 The mutations selected to increase stability may comprise one or more mutations selected from: R8H, A23S, V40I, K86M, K86E, I97V, P104Q, G135S, G135A, S143G, C157S, C159S, S174L, T178A, R190P, L191P, H212R, F217L, H219R, V231D, L233S, A235G, R236P, L265V, P270A, L296R, D304_D305Ins(DDYQVRSHSPVYC), I308A, K309R, E311A, Y312H, A319S ,I337L, H344N, and P350R. 15 Examples of methyltransferases engineered from SEQ ID NO: 19 include those of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. 20 In accordance with another possibility, the methyltransferase may have an amino acid sequence with at least 90 % sequence identity to SEQ ID NO: 20. An example of a methyltransferase engineered from SEQ ID NO: 20 has a sequence according to SEQ ID NO: 28. 25 Additional mutations introduced in such variants are not particularly limited provided that the methyltransferase remains capable of transferring a methyl group from the cofactor to the substrate. The additional mutations may comprise additions, deletions, or substitutions. Variants may include one or more conservative mutations. 30 Variants may include one or more mutations to improve binding of the substrate. For example, a histidine or tryptophan residue may be introduced at a position selected to provide a stacking interaction with the pseudouridine base. Alternatively or additionally, a residue such as K or R capable of forming a salt bridge to a phosphate group at position R1of the substrate may be introduced. Alternatively or additionally, substitutions may be made to improve hydrogen bonding to the ribose or deoxyribose group of the substrate. 5 Although the above methyltransferases have been described in the context of the method of synthesising a 1N-alkyl derivative of pseudouridine, it should be appreciated that the methyltransferases as such represent an independent aspect of the present disclosure. 10 Related aspects provide an isolated polynucleotide having a sequence encoding a methyltransferase as defined herein; an expression vector carrying the polynucleotide; and a microorganism transduced with the expression vector. The microorganism may be E. coli. Example Class IV (SPOUT) methyltransferases 15 The methyltransferase used in the method may be a dimeric SPOUT methyltransferase comprising a SPOUT domain configured to transfer the methyl group from the cofactor to the substrate. As will be demonstrated in the Examples, various SPOUT methyltransferases have been found to be useful for methylating pseudouridine derivatives of Formula 1. 20 The methyltransferase may be a minimalist dimeric SPOUT methyltransferase, free of any domains other than the SPOUT domain. Such a methyltransferase may optionally include a short terminal extension, e.g. an N-terminal extension, including no more than 50 amino acid residues. Preferably, the methyltransferase may consist of the SPOUT domain. 25 Typically, the SPOUT domain includes an amino acid residue which forms a stacking interaction with the substrate when the SPOUT domain binds to the substrate. This residue is referred to herein as a “stacking residue”. It has been found that wild-type SPOUT methyltransferases that provide a stacking interaction (e.g., proteins N1 and N2 as discussed 30 in the Examples) are useful in the methods described herein. SPOUT methyltransferases N3 and N4 that did not provide the stacking interaction had activities below the lower limit of detection. However, modifying these methyltransferases to provide the stacking interaction resulted in enzymes with good activity. The amino acid residue which forms the stacking interaction may be a histidine residue, a 5 tyrosine residue, or tryptophan residue, located proximal to the catalytically-active residues of the protein. In this context, “proximal” describes the relationship between the stacking amino acid residue and the catalytically-active residues in the quaternary structure of the dimeric SPOUT methyltransferase. The stacking and catalytically-active resides are proximal when the stacking interaction between the stacking residue and the substrate facilitates 10 catalysis of the alkylation reaction by the active residues. The mechanism of action of methyltransferases is well-characterized in the literature (see Krishnamohan and Jackman, Biochemistry.2019 February 05; 58(5): 336–345, in particular Fig. 3). The active site residues and nearby residues can be identified using routine 15 techniques. Alternatively or additionally, when group R1of the substrate is a monophosphate group, a diphosphate group, or a triphosphate group, the SPOUT domain may include an amino acid residue which forms a salt bridge with a phosphate group of the substrate when the SPOUT 20 domain binds to the substrate. The amino acid residue which forms the salt bridge may be a lysine or arginine residue. The methyltransferase may be a tRNA (pseudouridine54-N1)-methyltransferase. Two examples of tRNA (pseudouridine54-N1)-methyltransferases were investigated, and both 25 displayed a useful level of activity. It was further uncovered that activity could be improved by introducing various mutations. One example of a useful tRNA (pseudouridine54-N1)-methyltransferase has an amino acid sequence of SEQ ID NO: 1. Variants of this methyltransferase which include at least one 30 mutation and have at least 70 % sequence identity to SEQ ID NO: 1 are also contemplated. The amino acid sequence may include mutations K10Q, K75N, and either K20D or K20N. In such implementations, the amino acid sequence may further include one or more mutations selected from K74D, R81L, and K89N. Variants of SEQ ID NO: 1 containing these combinations of mutations were found to have improved activity. 5 Variants of SEQ ID NO: 1 may alternatively or additionally include one or more mutations selected from D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, I193V, K199R, K200R, R201E, C203F. 10 Variants of SEQ ID NO: 1 may alternatively or additionally include one or more mutations selected from D104Y, R115K, N121D, V123I, M138K, N139D, I155L, R167K, R176K, K200R, R201N, C203del, E204del, I205del. Variants of SEQ ID NO: 1 may alternatively or additionally include one or more mutations 15 selected from: L32V, D38S, Q59E, C65A, S71N, E100N, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, I154V, I155L, R167K, D170E, K173G, R176K, I177V, N187D, I193V, K200R, R201G, C203del, E204del, and I205del. 20 Variants of SEQ ID NO: 1 may alternatively or additionally include one or more mutations selected from K10Q, P23D, S77K, and D79N. Other examples of useful mutations to SEQ ID NO: 1 include P23K, S77D, and F41N. 25 Specific examples of useful methyltransferases include the following: i) SEQ ID NO: 1 with mutations K10Q, K20D, and K75N (i.e., Enzyme E1 as set out in the Examples, having a sequence of SEQ ID NO: 9); ii) SEQ ID NO: 1 with mutations K10Q, K20N, K75N, and R81L; iii) SEQ ID NO: 1 with mutations K10Q, K20D, K75N, K74D, and K89N; 30 iv) SEQ ID NO: 1 with mutations D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, I193V, K199R, K200R, R201E, and C203F (Enzyme E5, SEQ ID NO: 15); v) SEQ ID NO: 1 with mutations K10Q, P23D, S77K, and D79N (i.e., Enzyme E2, SEQ ID NO: 10); vi) SEQ ID NO: 1 with mutations D104Y, R115K, N121D, V123I, M138K, N139D, I155L, R167K, R176K, K200R, R201N, C203del, E204del,and I205del (Enzyme E12, SEQ ID NO: 5 13); vii) SEQ ID NO: 1 with mutations L32V, D38S, Q59E, C65A, S71N, E100N, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, I154V, I155L, R167K, D170E, K173G, R176K, I177V, N187D, I193V, 10 K200R, R201G, C203del, E204del, and I205del (Enzyme E7, SEQ ID NO: 14). Another example of a useful tRNA (pseudouridine54-N1)-methyltransferase has an amino acid sequence of SEQ ID NO: 2. Variants of this methyltransferase which include at least one mutation and have at least 70 % sequence identity to SEQ ID NO: 2 are also contemplated. 15 The mutation(s) present in the variants are not particularly limited provided that the methyltransferase remains capable of transferring a methyl group from the cofactor to the substrate. The additional mutations may comprise additions, deletions, or substitutions. For instance, variants may include one or more conservative mutations. 20 The methyltransferase may alternatively be an rRNA small subunit pseudouridine methyltransferase Nep1 engineered to methylate the substrate. The engineered rRNA small subunit pseudouridine methyltransferase Nep1 may comprise a mutation compared to a wild-type Nep1, the mutation introducing: 25 i) a stacking amino acid residue selected from a histidine residue, a tyrosine residue, and a tryptophan residue, the stacking amino acid residue being arranged proximal to one or more amino acid residues which catalyse transfer of the methyl group or ethyl group from the cofactor to the substrate; and / or ii) a salt bridging amino acid residue configured to form a salt bridge to a 30 phosphate group of the substrate, the salt bridging amino acid being selected from a lysine residue and an arginine residue, and the salt bridging amino acid being arranged proximal to one or more amino acid residues which catalyse transfer of the methyl group or ethyl group from the cofactor to the substrate. Mutations i) and / or ii) may improve substrate binding and may increase activity of the methyltransferase. 5 Alternatively or additionally, the engineered rRNA small subunit pseudouridine methyltransferase Nep1 may comprise iii) a mutation to the active site of the enzyme which replaces a negatively-charged amino acid residue (D or E) with a neutrally-charged amino acid residue selected from N, S, C, A, T, M, G, V, L, and I or a positively-charged amino acid residue 10 selected from R, K and H. The alkylation reaction proceeds via an intermediate in which H is removed from the N1 position of the pseudouridine base, and removing a negative charge or preferably replacing the negative charge with a positive charge at the active site may therefore improve enzyme activity. 15 Preferably, at least one but no more than two positively-charged residues are introduced at the active site. The rRNA small subunit pseudouridine methyltransferase Nep1 may have an amino acid sequence having 70 to 99.6 % sequence identity to SEQ ID NO:3. The amino acid sequence 20 has less than 100 % identity to SEQ ID NO:3, and in other words includes at least one mutation compared to SEQ ID NO: 3. The at least one mutation to SEQ ID NO: 3 may include V2_A27del. This mutation corresponds to the omission from the N-terminus a strand of 26 amino acids. Omitting this strand 25 improved the level of expression of the methyltransferase by a producer cell, as well as improving solubility. The at least one mutation to SEQ ID NO: 3 may comprise at least one of K106Q, R129N, and S233W. 30 The at least one mutation to SEQ ID NO: 3 may comprise S233W and optionally one or more of L97A, D101A, K106Q, R129N and R132S. The at least one mutation may comprise S233W, V2_A27del, and optionally one or more of L97A, D101A, K106Q, R129N and R132S. The at least one mutation may comprise S223W; one or more of L97A, D101A, and R132S; and optionally V2_A27del. 5 Further variants may include additional mutations, e.g. conservative mutations, provided that the methyltransferase remains capable of transferring a methyl group from the cofactor to the substrate. Provided are the following methyltransferases: 10 i) SEQ ID NO: 3 with mutations S223W, L97A, D101A, and R132S; and ii) SEQ ID NO: 3 with mutations V2_A27del, S223W, L97A, D101A, and R132S. A further example of an rRNA small subunit pseudouridine methyltransferase Nep1 useful in the methods described herein is a methyltransferase having an amino acid sequence having 15 70 to 99.6 % sequence identity to SEQ ID NO: 4. The amino acid sequence has less than 100 % identity to SEQ ID NO: 4, and in other words includes at least one mutation. The at least one mutation may comprise D210H, D210Y, or D210W. These mutations introduce stacking interactions with the pseudouridine base. The mutations may further 20 comprise R107S, particularly in implementations which include D210H. Alternatively or additionally, the at least one mutation may comprise W212A, W212H, or W212N. 25 The at least one mutation may comprise: a) D210H and W212H; or b) D210Y and W212N. The mutations may further comprise I45K and optionally Y49E. 30 The mutations may further comprise G76A or G76N. The mutations may comprise D210Y and Q186A. The mutations may comprise one or more of K104N, R111Q, and K187H. 5 The mutations may include one or more of R107S and Q186A. In a more specific example, there is provided a methyltransferase having an amino acid sequence of SEQ ID NO: 4 with mutations I45K, Y49E, G76A, K104N, R107S, R111Q, Q186A, K187H, D210Y, and W212N. 10 Another example methyltransferase has an amino acid sequence of SEQ ID NO: 4 with mutations I45K, Y49E, G76A, K104N, R107S, R111Q, Q186A, K187H, D210W, and W212N. Another example methyltransferase has an amino acid sequence of SEQ ID NO: 12. 15 Although the above methyltransferases have been described in the context of the method of synthesising a 1N-alkyl derivative of pseudouridine, it should be appreciated that the methyltransferases as such represent an independent aspect of the present disclosure. 20 Related aspects provide an isolated polynucleotide having a sequence encoding a methyltransferase as defined herein; an expression vector carrying the polynucleotide; and a microorganism transduced with the expression vector. Preparing the substrate 25 The methods provided herein may further comprise preparing the substrate from a precusor. Pseudouridine 5’-monophosphate may be formed by contacting D-ribose-5-monophosphate with uracil and a pseudouridine monophosphate glycosidase. The pseudouridine 5’- 30 monophosphate may be used directly as a substrate for the methyltransferase. Alternatively, the pseudouridine 5’-monophosphate may be converted to pseudouridine using a phosphatase. The D-ribose-5-monophosphate may be formed by contacting α-D-ribose-1-monophosphate with a phosphopentomutase. The α-D-ribose-1-monophosphate may be formed by contacting uridine with a uridine phosphorylase and an inorganic phosphate. 5 The substrate may conveniently be prepared in vivo. Conveniently, both the preparation of the substrate and the synthesis of the 1N-alkyl derivative of pseudouridine may be performed in vivo using the same microorganism. The microorganism may be engineered to express the pseudouridine monophosphate glycosidase, phosphopentomutase, uridine phosphorylase, 10 and the methyltransferase. Pseudouridine-5' -monophosphate may in particular be prepared by the method described in WO 2023 / 131727 A1. 15 Examples Example 1. SPOUT methyltransferases Enzymes potentially capable of methylating pseudouridine substrates were identified by 20 searching sequence databases for methyltransferase sequences similar to known methyltransferases that were shown to methylate pseudouridine bases in RNA sequence context. Structural models were predicted, and structural diversity evaluated. The sequences were then grouped into clusters. Representative members of each of the clusters were investigated by modelling interactions between the protein and the desired substrates. 25 Enzymes predicted to alkylate pseudouridine nucleosides or nucleotides were selected for testing. Cloned genes of identified enzymes were synthesized (Twist Bioscience). Plasmids carrying the genes were transformed into E. coli using electroporation, in which electro-competent E. 30 coli were subjected to 18,000 V / cm. A 5'-methylthioadenosine / S-adenosylhomocysteine nucleosidase-deficient strain of E. coli was used to avoid S-adenosylhomocysteine (SAH) degradation. Expression was carried out in media consisting of 2 % tryptone (Formedium), 1 % yeast extract (Formedium), 2 % NaCl (Roth) and 100 µg / mL ampicillin (Sigma-Aldrich) using T7 RNA polymerase / promoter system. The cells were grown at 37 °C, induced with 0.5 mM IPTG 5 (Sigma-Aldrich) and 0.1 % L-rhamnose (Roth) at OD 600 = 0.8 and grown for 22 h at 16 °C afterwards. The proteins were expressed with 6 C-terminal histidine residues to facilitate purification. Multiple purified enzymes produced in this way were tested for their ability to methylate pseudouridine Ψ, pseudourdine monophosphate ΨMP, and pseudouridine triphosphate ΨTP. 10 The enzymes tested are discussed further below. Optimised buffer conditions for enzymatic production of N1-methylpseudouridine and its derivatives were identified by performing reactions in various 25 mM Tris-HCl solutions 15 having different pHs, NaCl concentrations, and MgCl2concentrations. Each solution included 100 mM KCl, 25 μM S-adenosyl-L-methionine (SAM), 100 μM pseudouridine-5’- monophosphate or pseudouridine-5’-triphosphate as a substrate, and 0.7 to 9 μM methyltransferase in a total volume of 25 µL (Table 1). Reaction mixtures were incubated at 37 °C for 24 h and monitored by detecting S-adenosylhomocysteine (SAH), a secondary 20 product of methylation. SAH was detected using a commercial kit which quantifies SAH through an enzyme-coupled reaction with bioluminescent readout (MTase-Glo™ Methyltransferase Assay, Promega). SAH concentrations were determined according to the calibration curve. Buffer # pH [NaCl] / [MgCl2] / [KCl] / [SAM] / Substrate / mM mM mM µM µM 1 7.5 0 10 100 25 100 2 8.5 0 1 100 25 100 3 7.5 100 0 100 25 100 4 8.5 100 10 100 25 100 5 8.5 300 10 100 25 100 6 7.5 300 1 100 25 100 7 8.5 300 0 100 25 100 8 7.5 300 10 100 25 100 The table above identifies the buffer solutions used, and Fig.2 shows the amount of SAH detected in each reaction mixture. Buffer 6, comprising 25 mM Tris-HCl at pH 7.5, 100 mM KCl, 300 mM NaCl, 1mM MgCl2 gave the best performance. These conditions with slight 5 modifications were used in further experiments. Methylation activity was tested in a reaction mixture containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 300 mM NaCl, 1mM MgCl2, 0.03 % NP-40, 25 µM S-adenosyl-L-methionine (SAM), 1 mM of substrate (Ψ, ΨMP or ΨTP) and 2.5 to 20 µM methyltransferase (depending on the 10 protein used), in a total volume of 25 µL. The reaction mixture was incubated at 37 °C for 24 h, at which point the reaction was stopped. Methylation activity was determined by measuring the amount of S-adenosylhomocysteine (SAH) present in the reaction mixture. SAH is a secondary product of methylation. SAH was 15 detected using a commercially-available kit (MTase-Glo™ Methyltransferase Assay, Promega). By way of illustration, Fig.3 shows that SAH was detected in the above-described assay when using protein N1 as the methyltransferase with Ψ, ΨMP, and ΨTP as substrates. Only a trace 20 amount of SAH was detected in the absence of any substrate. HPLC-MS was used to confirm that the products of the reactions were 1N-methyl-Ψ or its phosphorylated derivatives. For the HPLC-MS (ESI) analysis, samples containing phosphorylated Ψ nucleotides were first treated with alkaline phosphatase to remove the 25 phosphate. Two ion transitions were monitored to detect Ψ in the analysed samples: 259.1 [M+H]+^ 139.1 and 259.1 [M+H]+^ 169.1. Peaks with retention times in the range 3.4 to 3.629 min had the above-mentioned m / z values. 5 Figs.4A to 4C are mass chromatograms of reaction mixtures prepared using protein N1, after treatment with alkaline phosphatase. The chromatograms show ion transitions 259.1 [M+H]+ 169.1 with retention times of 3.4 to 3.629 min, which correspond to 1N-methylpseudouridine. Fig.4A shows results obtained using pseudouridine as the substrate; Fig.4B shows results obtained using pseudouridine-5’-monophosphate as a 10 substrate; and Fig. 4C shows results obtained using pseudouridine-5’-triphosphate as a substrate. Analogous chromatograms were observed for others of the tested enzymes. For comparison, Fig.5 shows a mass chromatograph of a commercially-obtained standard sample of 1N-methyl-Ψ. As may be seen, the retention time and 258.0852 Da molar mass of 15 the standard sample closely match those of the product of the present method. The slight difference in retention time is believed to be the result of the presence of additional components in the reaction mixture. The identity of the produced substance was further confirmed by spiking reaction mixtures 20 with commercially available standard and observing the area increase of target peaks for ion m / z 139.1 (ion transition 259.1 139.1) and 169.1 (ion transition 259.1 [M+H]+ 169.1). The results of tests performed using enzyme N1 are shown in Figs.6A to 6C, and analogous chromatograms were observed for other identified enzymes and other substrates. Fig.6A shows the mass chromatogram of the unspiked reaction mixture; Fig.6B shows the 25 mass chromatogram of the spiked reaction mixture; and Fig.6C is an overlayed view of Figs. 6A and 6B. The enzymes that were active with pseudouridine and / or its derivatives include both natural and engineered proteins with sequence identity levels as low as 16 %. This is illustrated in Fig. 30 7, which is a pairwise identity matrix showing levels of sequence identity amongst 6 illustrative SPOUT methyltransferases that were found to be active using assays as described above. In order to measure reaction yield, samples were treated with alkaline phosphatase and analysed with HPLC-MS. The amount of 1N-methyl-Ψ in the sample was calculated by comparing area of 1N-methyl-Ψ peak in the measured samples to the area of 1N-methyl-Ψ 5 peak in a standard samples with known concentration. In the most efficient reaction of those performed in this study, the yields of 1N-methyl-Ψ, 1N-methyl-ΨMP and 1N-methyl-ΨTP were approximately 37 %; 36 % and 37 % respectively. The table below shows the measured level of activity of selected enzymes. The list of enzymes 10 is not exhaustive. At least 42 further enzymes which were active on some or all of the substrates were identified in the present study. AcƟvity on Substrate Protein (SEQ ID NO) Ψ ΨMP ΨTP N1 (SEQ ID NO: 1) +++ ++ ++ N2 (SEQ ID NO: 2) n.d. ++ +++ E1 (SEQ ID NO: 9) +++ + ++ E2 (SEQ ID NO: 10) +++ ++ ++ E3 (SEQ ID NO: 11) ++ ++ +++ E4 (SEQ ID NO: 12) +++ +++ +++ Key: 15 N: natural enzyme E: engineered enzyme n.d: not determined +: low relative activity ++: medium relative activity 20 +++: high relative activity Relative activity scales were different for each enzyme. Various ones of the enzymes tested are discussed in detail below. Protein N1 Protein N1 is a tRNA (pseudouridine54-N1) methyltransferase derived from 5 Methanocaldococcus jannaschii. It is a dimeric protein, belonging to the SPOUT superfamily of SAM-dependent methyltransferases. The dimer includes an α / β knot structural domain that forms part of the SAM binding pocket. N1 has an amino acid sequence of: 10 MREFIFKANKTITSSDINLKDLPGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCIK FVGSELKKVSPDERNIAIFIKKALKKFEELDEEQRKDWNQSTPGIYVRRLGFRNLVLEKLEEGKNIY YLHMNGEDVENVDIENPVFIIGDHIGIGEEDERFLDEIKAKRISLSPLELHANHCITIIHNVLDKKRI CEI (SEQ ID NO: 1) 15 Residues R28, D30, and R34 were identified as active site residues. Residue H185 was identified as a substrate binding residue that forms a stacking interaction with the pseudouridine base. 20 The following mutations to SEQ ID NO: 1 were identified as potentially beneficial: MutaƟon ObservaƟons K10Q Improves acƟvity when combined with K20D, K75N and opƟonally (i) R81L or (ii) K74D and K89N. K10Q alone did not result in a measurable change in acƟvity. It is believed that K10 pulls the substrate away from the catalyƟc site, and replacing K10 with another residue is therefore advantageous. F41N Increases affinity for pseudouridine by hydrogen bonding to the ribose sugar. S77K Increases affinity for phosphorylated pseudouridine derivaƟves by forming a salt bridge to the phosphate group. S77R Increases affinity for phosphorylated pseudouridine derivaƟves by forming a salt bridge to the phosphate group. S77K provides a larger improvement. P23D Improves orientaƟon of posiƟve charge from posiƟon 77 but potenƟally reduces protein solubility. P23K Increases affinity for phosphorylated pseudouridine derivaƟves by forming a salt bridge to the phosphate group. S77D Useful in combinaƟon with P23K. May orient P23K in place. The following mutants of protein N1 were investigated: ID MutaƟons ObservaƟons E1 K10Q, K20D, K75N Improvement in acƟvity compared to WT E2 K10Q, P23D, S77K, D79N Improvement in acƟvity compared to WT. More acƟve than N2. E9 K10Q, K20N, K75N, R81L Improvement in acƟvity compared to WT E10 K10Q, K20D, K75N, K74D, K89N Improvement in acƟvity compared to WT E11 K10Q Approximately the same the acƟvity as WT E5 D38S, C65A, I66L, Q107E, N145E, Most acƟve mutant of SEQ ID 1 out of E149K, I155L, R176K, I193V, those tested. K199R, K200R, R201E, C203F E12 D104Y, R115K, N121D, V123I, Second most acƟve mutant of SEQ ID NO: M138K, N139D, I155L, R167K, 1 R176K, K200R, R201N, C203del, E204del,and I205del E7 L32V, D38S, Q59E, C65A, S71N, Third most acƟve variant of SEQ ID NO: 1 E100N, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, I154V, I155L, R167K, D170E, K173G, R176K, I177V, N187D, I193V, K200R, R201G, C203del, E204del, and I205del E1 had the following sequence: MREFIFKANQTITSSDINLDDLPGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCI KFVGSELKNVSPDERNIAIFIKKALKKFEELDEEQRKDWNQSTPGIYVRRLGFRNLVLEKLEEGKNI 5 YYLHMNGEDVENVDIENPVFIIGDHIGIGEEDERFLDEIKAKRISLSPLELHANHCITIIHNVLDKKR ICEI (SEQ ID NO: 9) E2 had the following sequence: MREFIFKANQTITSSDINLKDLDGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCI 10 KFVGSELKKVKPNERNIAIFIKKALKKFEELDEEQRKDWNQSTPGIYVRRLGFRNLVLEKLEEGKNI YYLHMNGEDVENVDIENPVFIIGDHIGIGEEDERFLDEIKAKRISLSPLELHANHCITIIHNVLDKKR ICEI (SEQ ID NO: 10) E5 had the following sequence: 15 MREFIFKANKTITSSDINLKDLPGSCGRLDLLCRCVSSAFFLSHDIRRDVVFYAVLYGQPNPPVALK FVGSELKKVSPDERNIAIFIKKALKKFEELDEEQRKDWNESTPGIYVRRLGFRNLVLEKLEEGKNIY YLHMNGEDVEEVDIKNPVFILGDHIGIGEEDERFLDEIKAKKISLSPLELHANHCITIVHNVLDRREI FEI (SEQ ID NO: 15) E12 had the following sequence: MREFIFKANKTITSSDINLKDLPGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCIK FVGSELKKVSPDERNIAIFIKKALKKFEELDEEQRKYWNQSTPGIYVKRLGFRDLILEKLEEGKNIYY LHKDGEDVENVDIENPVFILGDHIGIGEEDEKFLDEIKAKKISLSPLELHANHCITIIHNVLDKRNI 5 (SEQ ID NO: 13) E7 had the following sequence: MREFIFKANKTITSSDINLKDLPGSCGRLDLVCRCVSSAFFLSHDIRRDVVFYAVLYGEPNPPVAIK FVGNELKKVSPDERNIAIFIKKALKKFEELDENKREEWTESTPGIYVSKKGFRDLILEKKKEGKKIYYL 10 HKDGEDIENVKINENENPVFVLGDHIGIGEEDEKFLEEIGAKKVSLSPLELHADHCITIVHNVLDKR GI (SEQ ID NO: 14) Protein N2 15 Protein N2 is a tRNA (pseudouridine54-N1)-methyltransferase derived from Thermococci archaeon. N2 is a dimeric protein, belonging to the SPOUT superfamily of SAM-dependent methyltransferases. The dimer includes an α / β knot structural domain that forms part of the SAM binding pocket. 20 Protein N2 has an amino acid sequence of: MVVFIVKSNTAKTKFLLKDLPGSGKRIDILCRCVNSAFCLSHDIRKDVILYLCFAGKTIKFVGKELKH LTPDERGIAILIRKALEGNPTPGVYVSEKSFQDTLLESGKEIIYLDERGEDISHLKLKKDMCFVLGDH LGFDREDQKILEKIGKKISISPKILHADHCIIVVHNFLDRS (SEQ ID NO: 2) 25 Residues R26, D28, and R32 were identified as active site residues. Residue H126 was identified as a substrate binding residue that forms a stacking interaction with the pseudouridine base. Protein N3 Protein N3 is rRNA small subunit pseudouridine methyltransferase Nep1 from Saccharomyces cerevisiae. N3 is a dimeric protein belonging to the SPOUT superfamily of SAM-dependent 5 methyltransferases. The dimer includes an α / β knot structural domain that forms part of the SAM binding pocket. Protein N3 has an amino acid sequence of: MVEDSRVRDALKGGDQKALPASLVPQAPPVLTSKDKITKRMIVVLAMASLETHKISSNGPGGD 10 KYVLLNCDDHQGLLKKMGRDISEARPDITHQCLLTLLDSPINKAGKLQVYIQTSRGILIEVNPTVRI PRTFKRFSGLMVQLLHKLSIRSVNSEEKLLKVIKNPITDHLPTKCRKVTLSFDAPVIRVQDYIEKLDD DESICVFVGAMARGKDNFADEYVDEKVGLSNYPLSASVACSKFCHGAEDAWNIL (SEQ ID NO: 3) 15 Residues R88, D90, and R132 were identified as active site residues. R136 - potentially forms a salt bridge with the psUMP / psUTP phosphate Removal of the 26 underlined amino acids from N-terminus was found to improve expression 20 and solubility of the protein. This removal corresponds to a mutation of V2_A27del. The following mutations to SEQ ID NO: 3 were identified as beneficial. Position numbering is based on wild-type, non-truncated, protein N3. MutaƟon ObservaƟons K106Q Reduces non-producƟve binding of substrate. R129N Forms a hydrogen bond to the ribose group of the substrate. S233W Improves substrate binding by forming a stacking interacƟon with the pseudouridine base. R132S Makes space for S233W and helps to orient catalyƟc residue R88. L97A Makes space for S233W. D101A Makes space for S233W. The most active mutant of SEQ ID NO: 3 which was tested, protein E3, contained the following four mutations: L97A, D101A, R132S, and S233W. The sequence of E3 was: MVEDSRVRDALKGGDQKALPASLVPQAPPVLTSKDKITKRMIVVLAMASLETHKISSNGPGGDK 5 YVLLNCDDHQGLLKKMGRDISEARPDITHQCLATLLASPINKAGKLQVYIQTSRGILIEVNPTVRIP STFKRFSGLMVQLLHKLSIRSVNSEEKLLKVIKNPITDHLPTKCRKVTLSFDAPVIRVQDYIEKLDDD ESICVFVGAMARGKDNFADEYVDEKVGLSNYPLWASVACSKFCHGAEDAWNIL (SEQ ID NO: 11) 10 Protein N4 Protein N4 is an rRNA small subunit pseudouridine methyltransferase Nep1 from Candidatus Verstraetearchaeota archaeon. N4 is a dimeric protein belonging to the SPOUT superfamily of SAM-dependent methyltransferases. The dimer includes an α / β knot structural domain 15 that forms part of the SAM binding pocket. Protein N4 has an amino acid sequence of: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDISLHYEAMKKLPNFEKRGRPDII HTTLLTILGSPANLEGLVRTYIHTINDQVVYIDPSVKIPRNYNRFVGLMEQLLKEGRVPPKGDLVL 20 MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFQKGSFLKKHVELADK VYAVYGSPLDTWVIASMLLHGYEIEKGII (SEQ ID NO: 4) Residues R63, D65, and R107 were identified as active site residues. 25 The following mutations to SEQ ID NO: 4 were identified as beneficial: MutaƟon ObservaƟons D210H Improves substrate affinity by forming a stacking interacƟon with the pseudouridine base when combined with a further mutaƟon to provide space for this interacƟon (e.g., R107S and / or W212H). D210W Improves substrate affinity by forming a stacking interacƟon with the pseudouridine base. D210Y Improves substrate affinity by forming a stacking interacƟon with the pseudouridine base. Provides a larger improvement in acƟvity than D210H or D210W. R107S Makes space for stacking from D210 posiƟon and helps to orient R63 by hydrogen bonding to D65. W212A Makes space for stacking from D210 posiƟon. W212H Makes space for stacking from D210 posiƟon. Provided a greater improvement than W212A for mutants containing D210H. W212N Makes space for stacking from D210 posiƟon. Provided a greater improvement than W212A for mutants containing D210Y. I45K May improve affinity for phosphorylated substrates by forming a salt bridge. Provided liƩle effect on acƟvity when used alone. A synergisƟc improvement in acƟvity was observed when this mutaƟon was combined with D210H, D210W, or D210Y. Y49E Helps to orient I45K. K104N May hydrogen bond to the ribose moiety of the substrate. R111Q May reduce non-producƟve binding. K187H May reduce non-producƟve binding. Q186A Increases acƟvity when combined with D210Y. G76A / N A G76A or G76N mutaƟon was present in all of the variants that included a mutaƟon at posiƟon D210. However, the effect of G76A / N was not enƟrely clear, and this mutaƟon is not believed to be essenƟal. Other possible mutations of SEQ ID NO: 4 include those selected from: L131, V132L, L133V, M134L, Y135M, V136Y, K137V, Q139K, S140Q, L141S, E142, G143L, L144E, L145G, K146L, E147L, I148K, K149E, P150I, T151K, R152P, F154R, M155T, L156F, S157M, E158L, N159S, G160E, E161N, K162G, I163E, N164K, S166N, T167I, L168S, A169T, K170L, E171A, L172K, 5 C173E, Q174L, E175C, N176Q, R177E, P178N, T179R, V180P, I181T, I182V, G183I, G184I, F185G, Q186G, K187F, G188Q, S189H, F190G, L191S, K192F, K193L, H194K, V195K, E196H, L197V, A198E, D199L, K200A, V201D, Y202K, A203V, V204Y, Y205A, G206V, S207Y, P208G, L209S, D210P, T211L, V213T, I214A, A215V, S216I, M217A, L218S, L219M, H220L, G221L, Y222H, E223G, I224Y, K226I, G227E, I228K, and I229G. 10 Of the variants of protein N4 tested, E6 was found to have the highest activity. E6 had the following combination of mutations: K104N, R111Q, K187H, D210Y, W212N, R107S, G76A, I45K, Y49E, and Q186A. The sequence of E6 is: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDKSLHEEAMKKLPNFEKRGRPDI 15 IHTTLLTILASPANLEGLVRTYIHTINDQVVYIDPSVNIPSNYNQFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFAHGSFLKKHVELADK VYAVYGSPLYTNVIASMLLHGYEIEKGII (SEQ ID NO: 16) Another engineered methyltransferase with an amino acid sequence similar to that of N4 was 20 E8, as set out hereinbelow (SEQ ID NO: 17). E8 can be expressed as SEQ ID NO: 4 with the following mutations: I45K, Y49E, G76A, K104N, R107S, R111Q, Q186A, K187H, D210W, and W212N. Another example of an engineered methyltransferase based on N4 is E4. E4 had the following 25 amino acid sequence: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDKSLHEEAMKKLPNFEKRGRPDI IHTTLLTILASPANLEGLVRTYIHTINDQVVYIDPSVNIPSNYNQFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFQHGSFLKKHVELADK VYAVYGSPLH TAVIASMLLH GYEIEKGII (SEQ ID NO: 12) 30 Compared to SEQ ID NO: 4, E4 includes the following mutations: I45K, Y49E, G76A, K104N, R107S, R111Q, L131, V132L, L133V, M134L, Y135M, V136Y, K137V, Q139K, S140Q, L141S, E142, G143L, L144E, L145G, K146L, E147L, I148K, K149E, P150I, T151K, R152P, F154R, M155T, L156F, S157M, E158L, N159S, G160E, E161N, K162G, I163E, N164K, S166N, T167I, L168S, A169T, K170L, E171A, L172K, C173E, Q174L, E175C, N176Q, R177E, P178N, T179R, V180P, I181T, I182V, G183I, G184I, F185G, Q186G, K187F, G188Q, S189H, F190G, L191S, K192F, 5 K193L, H194K, V195K, E196H, L197V, A198E, D199L, K200A, V201D, Y202K, A203V, V204Y, Y205A, G206V, S207Y, P208G, L209S, D210P, T211L, W212H, V213T, I214A, A215V, S216I, M217A, L218S, L219M, H220L, G221L, Y222H, E223G, I224Y, K226I, G227E, I228K, and I229G. Interestingly, E4 had a sequence similar to that of a wild-type SPOUT methyltransferase 10 derived from an unidentified microorganism believed to be in the phylum Verstraetearchaeota, having a sequence of: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDISLHYEAMKKLPNFEKRGRPDII HTTLLTILGSPANLEGLVRTYIHTINDQVVYIDPSVKIPRNYNRFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFQKGSFLKKHVELADK 15 VYAVYGSPLDTWVIASMLLHGYEIEKGII (SEQ ID NO: 18) E4 has the following differences with respect to SEQ ID NO: 18: I45K, Y49E, G76A, K104N, R107S, R111Q, K187H, V201, Y202V, A203Y, V204A, Y205V, G206Y, S207G, P208S, L209P, D210L, T211H, W212, V213T, I214A, A215V, S216I, M217A, L218S, L219M, H220L, G221L, 20 Y222H, E223, I224G, E225Y, K226E, G227I, I228E, and I229K. Accordingly, there is provided a methyltransferase having an amino acid sequence of SEQ ID NO: 18 with at least one, and preferably all, of these mutations. in vivo 25 E. coli cells were transformed by recombinant plasmids harbouring N1, N2, E3 enzymes or catalytic N1 mutant (negative control) by electroporation. Transformants were grown overnight at 37 °C on LB-agar plates (50 µg / mL ampicillin), suspended in 20 mL liquid LB- medium (50 µg / mL ampicillin) and grown in a shaker at 37°C until OD600 reached 0.6 – 0.8. 30 After reaching the desired OD600, flasks were put on ice for 30 min. IPTG and L-rhamnose were added to final concentrations of 0.5 mM and 0.1 %, respectively. The flasks were shaken at 16 °C for approximately 20 h.1.5 mL of each cell culture was collected by centrifugation, washed with 1 mL of 50 mM potassium phosphate buffer (pH 7.5) and resuspended into 100 µL of the same buffer that contained 5 mM of pseudouridine (Ψ). 5 Tubes containing reaction mixtures were incubated at 37 °C and 180 rpm for 3 days. The cells were separated from the buffer by centrifugation. The supernatant was mixed with acetonitrile (1:1) and analysed with HPLC-MS. The remaining cells were resuspended in 300 µL of 50 mM potassium phosphate buffer (pH 7.5) and lysed by sonication. The cell remnants were removed by centrifugation.5 µL of FastAP were added to 50 µL of each reaction mixture, 10 mixtures were incubated at 37 °C overnight.50 µL of acetonitrile was added and mixtures were centrifuged for 10 min at 4 °C at 16,000 rpm to precipitate the remaining proteins. The reaction mixtures were analysed by HPLC-MS. N1-methyl pseudouridine concentration was determined according to the calibration curve prepared from the standard. Results are 15 summarized in the table below. N1-methyl-Ψ yield, % N1-methyl-Ψ, µM N1 buffer 0.95 47.6 N1 cell content 0.083 4.16 N2 buffer 0.49 24.3 N2 cell content 0.01 0.48 E3 buffer 0.063 3.2 E3 cell content 0 0 Control buffer 0 0 Control cell content 0 0 The results demonstrate that N1-methyl pseudouridine can be produced in vivo using a microorganism that expresses a methyltransferase as provided herein. 20 Example 3. Enzymatic production of N1-methyl-2’-deoxypseudouridine and its derivatives Reaction mixtures containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 300 mM NaCl, 1mM MgCl2, 0.03 % NP-40, 25 μM S-adenosyl-L-methionine (SAM), 1 mM 2'-deoxypseudouridine 5 substrate (dΨ) and 7 μM N1 methyltransferase in a total volume of 25 µL were incubated at 37 °C. The reaction was stopped after 24 h and monitored by detecting S- adenosylhomocysteine (SAH), a secondary product of methylation. SAH was detected using a commercial kit which monitors the formation of SAH through an enzyme-coupled reaction with bioluminescent readout (MTase-Glo™ Methyltransferase Assay, Promega). The 10 measured SAH concentrations are shown in Fig.8. The data shown in Fig. 8 demonstrate that enzymes N1, E5, and E8 were all found to methylate dΨ. 15 In view of these results, active site similarities, structural modelling, and molecular docking data it is expected that SPOUT methyltransferases as described herein will methylate dΨ, dΨMP, and dΨTP. E8 had a sequence of: 20 MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDKSLHEEAMKKLPNFEKRGRPDI IHTTLLTILASPANLEGLVRTYIHTINDQVVYIDPSVNIPSNYNQFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFAHGSFLKKHVELADK VYAVYGSPLWTNVIASMLLHGYEIEKGII (SEQ ID NO: 17) 25 Example 4. Enzymatic production of N1-ethylpseudouridine and its derivatives Reaction mixtures containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 300 mM NaCl, 1mM MgCl2, 0.03 % NP-40, 20 μM S-adenosyl-L-ethionine (SAE), 1 mM pseudouridine substrate (Ψ or ΨMP) and 6-12 μM methyltransferase (according to the protein) in a total volume of 25 µL30 was incubated in 37 °C. The reaction was stopped after 24 h and monitored by detecting S- adenosylhomocysteine (SAH), a secondary product of ethylation (SAH was detected using a commercial kit which monitors the formation of SAH through an enzyme-coupled reaction with bioluminescent readout (MTase-Glo™ Methyltransferase Assay, Promega). SAH concentrations were determined according to the calibration curve, and are shown in Figs.9A (E5) and 9B (E6). 5 Figs.9A and 9B shows that E5 and E6 respectively can ethylate pseudouridine and derivatives thereof. Based on these results, and due to active site similarities, structural modelling, and molecular docking data it is expected that other SPOUT methyltransferases as described herein will also ethylate Ψ, ΨMP, ΨTP, dΨ, dΨMP or dΨTP. 10 Methyltransferases that have no reported activity with pseudouridine, but whose active sites are potentially capable of methylating pseudouridine substrates were identified as follows. 15 Known and putative methyltransferase sequences were collected from public and private databases. Structural models were predicted and structural diversity evaluated. The sequences were then grouped into clusters. Representative members of the clusters were investigated by modelling possible interactions between the protein and the desired substrates. 20 Genes encoding the identified enzymes and mutants thereof were synthesized (Twist Bioscience). Plasmids carrying the genes were transformed into E. coli using an electroporation process, in which electro-competent E. coli was subjected to 18,000 V / cm. A 5'-methylthioadenosine / S-adenosylhomocysteine nucleosidase-deficient strain of E. coli was 25 used to avoid S-adenosylhomocysteine (SAH) degradation. Expression was carried out in media consisting of 2 % tryptone (Formedium), 1 % yeast extract (Formedium), 2 % NaCl (Roth) and 100 µg / mL ampicillin (Sigma-Aldrich) using a T7 RNA polymerase / promoter system. The cells were grown at 37 °C, induced with 0.5 mM IPTG 30 (Sigma-Aldrich) and 0.1 % L-rhamnose (Roth) at OD 600 = 0.8 and grown for 22 h at 16 °C afterwards. The proteins were expressed with 6 C-terminal histidine residues to facilitate purification. Multiple purified enzymes produced in this way were tested for their ability to methylate natural substrate known from literature: guanosine-5'-triphosphate “GTP”. For this, reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 0.03 % NP40, 50 μM SAM, 5 mM 5 GTP and 0.3-12 μM enzyme (according to protein) were incubated at 37 °C. The reactions were stopped after 1 h and activity was assessed by detecting S-adenosylhomocysteine (SAH), a secondary product of methylation. SAH was detected using a commercial kit (MTase-Glo™ Methyltransferase Assay, Promega). The enzymes were found to be active on GTP native substrate. 10 Modelling has demonstrated that certain class I methyltransferases, specifically cap methyltransferases, would with small changes to their active site be capable of methylating pseudouridine and its derivatives. For example, modelling has shown that arginine residues correctly positioned in the active site will enable pseudouridine methylation. The methylation 15 reaction involves deprotonation of the pseudouridine base at the N1 position. The positive charge of the arginine residues will promote such deprotonation and / or stabilize the deprotonated base. Our experimental data with SPOUT methyltransferases and structural modelling of SPOUT 20 and Cap methyltransferases shows that Cap methyltransferases will be able to methylate and ethylate Ψ, ΨMP, ΨTP, dΨ, dΨMP, and dΨTP. Proteins S1 to S4 as discussed below were investigated. 25 Fig.10 is a pairwise sequence identity matrix of proteins S1 to S4. The Fig. demonstrates that, despite all being mRNA cap guanine-N7 methyltransferases, S1 to S4 have less than about 40 % sequence identity. Protein S1 Protein S1 is a Class 1 SAM dependent methyltransferase, more specifically an mRNA cap guanine-N7 methyltransferase from Nematocida parisii having an amino acid sequence of: 5 MEKSNNNVANHYNKIKSLGVQSREASKIIGVREANNFLKQKLIQKFIRENSVVLDLGCGKGGDLS KLKHHNIKHYYGCDIAKESLAEALKRSLTHKFKSDFLQADFINNKIIIQEKADLVMAQFSFHYAFA NENSVKKAVNNVCNNLKEGGVFILTIPDMQVITRRSARNIVDGSFGNSLYKVCPNKSFYKNELFG RGYEFHLQEALTGCEEYLIDLNYLTSHFASKGIKKIFDIDFLSFLNHEMSADKETYSRMVRHPLTKE ELPIIELYRAVAYKKNGS (SEQ ID NO: 5) 10 The activity of wild-type protein S1 was below the limit of detection of the assays used. However, analysis demonstrated that activity can be enhanced by introducing mutations. The following useful mutations to SEQ ID NO: 5 have been identified: MutaƟon Effect E212Q, E212N, E212S, Removal of negaƟve charge from posiƟon 212 should E212C, E212A, E212T, stabilize deprotonated pseudouridine base. E212M, E212H, E212G, E212V, E212L, or E212I. E212K The introducƟon of a posiƟve charge at posiƟon 212 should stabilize the deprotonated pseudouridine base more effecƟvely than removing the negaƟve charge. A K residue may downshiŌ the pKa of pseudouridine base. E212R Introducing an R residue at posiƟon 212 should be even more effecƟve than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases uƟlize catalyƟc arginine residues to downshiŌ the pKa of pseudouridine base. Y271R or Y217K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. F201R or F201K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. I267R or I267K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. Further mutations may be introduced, e.g. to improve the orientation of the R or K residues, thereby improving activity further. 5 Protein S2 Protein S2 is a Class 1 SAM dependent methyltransferase, more specifically an mRNA cap guanine-N7 methyltransferase from Catenaria anguillulae having an amino acid sequence of: MGDSATDQSRRAAAQLVANHYNQRQSSTVDSRKDSPIFHLRAFNNWVKAVLMNRFLRSGLH 10 LLDIGCGKGGDLNKYNKARIAQLYAFDVASVSIDQATERYRQMHRPWFKAQFQALDCYNDSIE PYMPRGASVGAVSMQFCAHYAFQSEKQVRIMLENVSRWLAPGGYYFGTVPDANVLVRKLRAS PGLEYGNSIYKIRFVQKDAYPVYGHEYSFLLEDAIDDCPEYLIHWPSFVRLAAEYGLEQVEHTNFH PFYHEQADKFRDLLVRMKVVTEDRPELSMDEWEAAGLYSVFVFRKRGS (SEQ ID NO: 6) 15 The activity of wild-type protein S2 was below the limit of detection of the assays used. However, analysis demonstrated that activity can be enhanced by introducing mutations. The following useful mutations to SEQ ID NO: 6 have been identified: MutaƟon Effect E226Q, E226N, E226S, Removal of negaƟve charge from posiƟon 226 should E226C, E226A, E226T, stabilize deprotonated pseudouridine base. E226M, E226H, E226G, E226V, E226L, or E226I. E226K The introducƟon of a posiƟve charge at posiƟon 226 should stabilize the deprotonated pseudouridine base more effecƟvely than removing the negaƟve charge. A K residue may downshiŌ the pKa of pseudouridine base. E226R Introducing an R residue at posiƟon 226 should be even more effecƟve than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases uƟlize catalyƟc arginine residues to downshiŌ the pKa of pseudouridine base. Y290R or Y290K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. F215R or F215K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. A286R or A286K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. Further mutations may be introduced, e.g. to improve the orientation of the R or K residues, thereby improving activity further. Protein S3 Protein S3 is a Class 1 SAM dependent methyltransferase, more specifically a mRNA cap guanine-N7 methyltransferase from Lachancea thermotolerans having an amino acid 5 sequence of: MSVVNVDQIIRKHYNERTFVAKRRRRHLSPIIKLRNFNNAIKYMLIDKFTFPGNVVLEMGCGKG GDLRKYGAAGISQFIGIDISNASIVEAQKRFSSMGNLDYQVILITGDCFGESLGVAVEPFPECRFPC DVVSAQFCLHYAFESEEKARRTLLNVTKSLKIGGYFIGTIPDSEFIRYKLNKITKDVDKPSWGNAIY KVTFENSDYQKNNNEFTSPFGQMYTYWLEDAIDNVPEYVIPFETLRSLADEYGLELELQMPFNA 10 FFVQEIPKWINKFSPKMQEGLQRSDGKYGVEGDEKEAASYFYTVFAFKKVKGS (SEQ ID NO: 7) The activity of wild-type protein S3 was below the limit of detection of the assays used. However, analysis demonstrated that activity can be enhanced by introducing mutations. 15 The following useful mutations to SEQ ID NO: 7 have been identified: MutaƟon Effect E235Q, E235N, E235S, Removal of negaƟve charge from posiƟon 235 should E235C, E235A, E235T, stabilize deprotonated pseudouridine base. E235M, E235H, E235G, E235V, E235L, or E235I. E235K The introducƟon of a posiƟve charge at posiƟon 235 should stabilize the deprotonated pseudouridine base more effecƟvely than removing the negaƟve charge. A K residue may downshiŌ the pKa of pseudouridine base. E235R Introducing an R residue at posiƟon 235 should be even more effecƟve than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases uƟlize catalyƟc arginine residues to downshiŌ the pKa of pseudouridine base. Y304R or Y304K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. Y224R or Y224K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. A299R or A299K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. Further mutations may be introduced, e.g. to improve the orientation of the R or K residues, thereby improving activity further. 5 Protein S4 Protein S3 is a Class 1 SAM dependent methyltransferase, more specifically an mRNA cap guanine-N7 methyltransferase from Lachancea thermotolerans having an amino acid sequence of: 10 MEGKKEEIREHYNSIRERGRESRQRSKTINIRNANNFIKACLIRLYTKRGDSVLDLGCGKGGDLLK YERAGIGEYYGVDIAEVSINDARVRARNMKRRFKVFFRAQDSYGRHMDLGKEFDVISSQFSFHY AFSTSESLDIAQRNIARHLRPGGYFIMTVPSRDVILERYKQGRMSNDFYKIELEKMEDVPMESVR EYRFTLLDSVNNCIEYFVDFTRMVDGFKRLGLSLVERKGFIDFYEDEGRRNPELSKKMGLGCLTRE ESEVVGIYEVVVFRKLVPESDA (SEQ ID NO: 8) 15 The activity of wild-type protein S4 was below the limit of detection of the assays used. However, analysis demonstrated that activity can be enhanced by introducing mutations. The following useful mutations to SEQ ID NO: 8 have been identified: MutaƟon Effect E210Q, E210N, E210S, Removal of negaƟve charge from posiƟon 210 should E210C, E210A, E210T, stabilize deprotonated pseudouridine base. E210M, E210H, E210G, E210V, E210L, or E210I. E210KThe introducƟon of a posiƟve charge at posiƟon 210 should stabilize the deprotonated pseudouridine base more effecƟvely than removing the negaƟve charge. A K residue may downshiŌ the pKa of pseudouridine base. E210R Introducing an R residue at posiƟon 210 should be even more effecƟve than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases uƟlize catalyƟc arginine residues to downshiŌ the pKa of pseudouridine base. Y269R or Y269K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. F199R or F199K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. V265R or V265K Introducing a posiƟve charge should stabilize the deprotonated pseudouridine base by downshiŌing the pKa of the pseudouridine base. Further mutations may be introduced, e.g. to improve the orientation of the R or K residues, thereby improving activity further. 5 Example 6. in vivo synthesis of 1N-methylpseudoridine from a uridine feedstock The methods of synthesising 1N-alkyl derivatives of pseudouridine described herein may be implemented in vivo. As an example, Fig.11 is a reaction scheme outlining the in vivo synthesis of 1N-methyl pseudouridine using uridine as a starting material. In the reaction scheme of Fig 11, uracil is first cleaved into uracil and ribose-1-monophosphate using uridine phosphorylase (udp) and inorganic phosphate. 5 A phosphopentomutase (deoB) then isomerizes the ribose-1-monophosphate into ribose-5- monophosphate. The ribose-5-monophosphate is then connected to uracil using pseudouridine monophosphate glycosidase (psuG), to obtain pseudouridine 5'-monophosphate. 10 In this example, since the reactions are done in vivo, cellular phosphatases dephosphorylate the pseudouridine monophosphate to form the corresponding nucleoside, pseudouridine. The pseudouridine is then methylated in accordance with the method provided herein, using 15 a suitable methyltransferases and S-adenosyl methionine (SAM) as the methyl group donor. This forms the 1N-methylpseudouridine. In a specific implementation of the above method, E. coli BL21 (DE3) competent cells were transformed by electroporation with two recombinant plasmids. The plasmids contained (i) 20 genes encoding enzymes for synthesising pseudouridine 5’-monophosphate synthesis from uridine (udp, deoB, and psuG), and (ii) a gene encoding an appropriate pseudouridine methyltransferase, respectively. Four such transformants having the following combinations of genes were investigated: i) pCDF-udp-deoB and pRSF-psuG-B2P3; 25 ii) pCDF-udp-deoB and pRSF-psuG-B2P23; iii) pCDF-udp-deoB and pRSF-psuG-B5P8; and iv) pCDF-udp-deoB and pRSF-psuG-B11P9. Transformants were grown overnight at 37 °C on LB-agar plates (50 µg / mL spectinomycin and 30 30 µg / mL kanamycin). Transformants were suspended in 20 mL liquid LB-medium (50 µg / mL spectinomycin and 30 µg / mL kanamycin) and grown in a shaker at 37 °C until OD600 reached 0.5–0.6. IPTG was added to a final concentration of 1 mM, and the flasks were shaken at 37 °C for 3 h. After induction was complete, 3 mL of each cell culture were collected by centrifugation, 5 washed with 1 mL of 125 mM potassium phosphate buffer (pH 7.0), and resuspended into 100 µL of the same buffer that contained 5 mM uridine and 0.5 mM MnCl2. Tubes containing these reaction mixtures were incubated at 37 °C and 300 rpm for 24 h. The cells were then separated from the buffer by centrifugation, resuspended in 300 µL of 10 125 mM potassium phosphate buffer (pH 7.0), and lysed by sonication. Cellular debris were removed by centrifugation.5 µL of FastAP were added to the tubes, that contained 50 µL of buffer in which reactions were done, or 50 µL of supernatant after cell sonication. The resulting mixtures were incubated at 37 °C and 300 rpm overnight.50 µL of acetonitrile were added to each tube, mixtures were centrifuged for 10 min at 4 °C at 16000 rpm to precipitate 15 the remaining proteins. The reaction mixtures were analyzed by HPLC-MS. The 1N-methyl pseudouridine (m1Ψ) concentration was determined according to the calibration curve prepared from the standard. N1-Methyl pseudouridine was detected in the reaction buffer where B2P3 20 methyltransferase was present, however no 1N-methyl pseudouridine was detected in analyzed cell content. The final yield of 1N-methyl pseudouridine was 0.162 %, corresponding to a 1N-methylpseudouriding concentration of 8.1 µM in the reaction mixture. 25 Enzymes potentially capable of methylating pseudouridine substrates were identified by searching sequence databases for methyltransferase sequences similar to known methyltransferases. Structural models were predicted, and structural diversity evaluated. The sequences were then grouped into clusters. Cluster representatives were investigated by 30 modelling possible interactions between the protein and the desired substrates. In the present study, two wild-type proteins, S5 and S6, were predicted to act on pseudouridine nucleosides or nucleotides. Protein S5 is a Class I methyltransferase, more specifically a xanthosine methyltransferase 5 from Coffea canephora having an amino acid sequence of: MELQEVLRMNGGEGDTSYAKNSAYNQLVLAKVKPVLEQCVRELLRANLPNINKCIKVADLGCA SGPNTLLTVRDIVQSIDKVGQEKKNELERPTIQIFLNDLFPNDFNSVFKLLPSFYRKLEKENGRKIG SCLIGAMPGSFYSRLFPEESMHFLHSCYCLQWLSQVPSGLVTESGISTNKGSIYSSKASRLPVQKA YLDQFTKDFTTFLRIHSEELFSHGRMLLTCICKGVELDARNAIDLLEMAINDLVVEGHLEEEKLDSF 10 NLPVYIPSAEEVKCIVEEEGSFEILYLETFKVLYDAGFSIDDEHIKAEYVASSVRAVYEPILASHFGEAI IPDIFHRFAKHAAKVLPLGKGFYNNLIISLAKKPEKSDV (SEQ ID NO: 19) Protein S6 is a Class I methyltransferase, and more specifically xanthosine methyltransferase from Coffea arabica having an amino acid sequence of: 15 MELQEVLRMNGGEGDTSYAKNSAYNHLVLNKVKPVLEQCIRELLRASLPNINKCIKVADLGCAS GPNTLLTVRDIVQSIDKVGQEKKNELERPTIQIFLNDLFQNDFNSVFKLLPSFYRKLEKENGRKIGS CLIGAMPGSFYSRLFPEESMHFLHSCYCLHWLSQVPSGLVTELGISTNKGSIYSSKASRLPVQKAY LDQFTKDFTTFLRIHSEELFSHGRMLLTCICKGVEFDALNAIDLLEMAINDLVVEGHLEEEKLDSFN LPVYIPSAEEVKCIVEEEGSFEILYLETFKVLYDAGFSIDDNYPVRSHVQVYSDEHIKAEYVASSVRA 20 VYEPILASHFGEAIIPDIFHRFAKHAAKVLPLGKAYYNNLIISLAKKPEKSDM (SEQ ID NO: 20) Protein S5 was modified by introducing mutations that were predicted to be activating and / or stabilizing to generate proteins S10, S11, S13, S16, S17, S18, and S19. Protein; SEQ ID MutaƟons compared to S5 (SEQ ID Engineering raƟonale NO: 19) S10 Q161H, A238S AcƟvaƟon. SEQ ID NO: 21 S11 R8H, A23S, V40I, K86E, P104Q, Protein stabilizaƟon. SEQ ID NO: 22 G135S, S143G, C159S, S174L, T178A, L191P S13 R8H, A23S, V40I, K86M, I97V, Protein stabilizaƟon. SEQ ID NO: 23 P104Q, G135A, S143G, C157S, C159S, S174L, T178A, R190P, L191P, H212R, F217L, H219R, V231D, L233S, A235G, R236P, L265V, P270A, L296R, D304_D305Ins(DDYQVRSHSPVYC), I308A, K309R, E311A, Y312H, A319S, I337L, H344N, P350R S16 R8H, A23S, V40I, K86E, P104Q, AcƟvaƟon of stabilized protein SEQ ID NO: 24 G135S, S143G, C159S, S174L, S11. A238S introduces T178A, L191P, A238S hydrogen bond with Ψ ribose. S17 R8H, A23S, V40I, K86E, P104Q, AcƟvaƟon of stabilized protein SEQ ID NO: 25 G135S, S143G, C159S, Q161H, S11. Q161H introduces S174L, T178A, L191P, , A238S hydrogen bond donor for O2 of Ψ base. S18 R8H, A23S, V40I, K86M, I97V, AcƟvaƟon of stabilized protein SEQ ID NO: 26 P104Q, G135A, S143G, C157S, S13 by the introducƟon of C159S, S174L, T178A, R190P, L191P, mutaƟon A238S. H212R, F217L, H219R, V231D, L233S, A235G, R236P, A238S, L265V, P270A, L296R, D304_D305insDDYQVRSHSPVYC, I308A, K309R, E311A, Y312H, A319S, I337L, H344N, P350R S19 R8H, A23S, V40I, K86M, I97V, AcƟvaƟon of stabilized protein SEQ ID NO: 27 P104Q, G135A, S143G, C157S, S13 by the introducƟon of C159S, S174L, T178A, R190P, L191P, mutaƟons Q161H and A238S. H212R, F217L, H219R, V231D, L233S, A235G, R236P, L265V, P270A, L296R, D304_D305insDDYQVRSHSPVYC, I308A, K309R, E311A, Y312H, A319S, I337L, H344N, P350R, Q161H, A238S The following variant of protein S6 was also generated: Protein; SEQ ID MutaƟons compared to S6 (SEQ ID Engineering raƟonale NO: 20) S20 A238S AcƟvaƟon. SEQ ID NO: 28 5 Cloned genes of the identified enzymes were synthesized (Twist Bioscience). Plasmids carrying these genes were transformed into E. coli using electroporation: electro-competent E. coli was subjected to 18,000 V / cm. A 5'-methylthioadenosine / S-adenosylhomocysteine nucleosidase-deficient strain of E. coli was used to avoid S-adenosylhomocysteine (SAH) degradation. Expression was carried out in media comprising 2 % tryptone (Formedium), 1 % 10 yeast extract (Formedium), 2 % NaCl (Roth) and 100 µg / mL ampicillin (Sigma-Aldrich) using a T7 RNA polymerase / promoter system. The cells were grown at 37 °C, induced with 0.5 mM IPTG (Sigma-Aldrich) and 0.1 % L-rhamnose (Roth) at OD 600 = 0.8, and then incubated for 24 h at 16 °C. The proteins were expressed with 6 C-terminal histidine residues to facilitate purification. Fig.12 shows photographs of gels used to purify the proteins, in which M is a 15 protein molecular weight ladder and the arrows indicate the positions of purified proteins. The activity of proteins S5, S6, S10, S11, S13, S16, S17, S18, S19 and S20 was determined as follows. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1mM MgCl2, 0.1 mM DTT (dithiothreitol), 0.03 % NP-40 (a non-ionic surfactant), 100 μM S-adenosyl-L- methionine (SAM), 2.5 mM pseudouridine substrate (Ψ) and 0.89 to 1.05 μM 5 methyltransferase (depending on the protein used) in a total volume of 25 μL were incubated at 30 °C. The reactions were stopped after 24 h and monitored by detecting S-adenosyl-L- methionine (SAH), a secondary product of methylation. SAH was detected using a commercial kit which monitors the formation of SAH through an enzyme-coupled reaction with bioluminescent readout (MTase-Glo™ Methyltransferase Assay, Promega). SAH 10 concentrations were determined according to a calibration curve. A bar chart showing the amount of SAH formed in each assay is provided as Fig.13. Proteins S6, S10, S11, S13, S16, S17, S18, S19 and S20 were all found to be active, with S16 to S20 having high levels of activity. 15 The identity of reaction product (synthesized N1-methyl-Ψ) and proof of enzyme specificity were confirmed by HPLC-MS / MS. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1mM MgCl2, 0.1 mM DTT, 0.03 % NP-40, 100 μM S-adenosyl-L-methionine (SAM), 2.5 mM pseudouridine substrate (Ψ) and 0.44-2.96 μM methyltransferase (according to 20 protein used) in a total volume of 30 µL were incubated in 30 °C for 24h. HPLC-MS / MS samples were prepared by diluting 3 μL of reaction mixture with 9 μL of MilliQ water and 12.5 µL acetonitrile. To denature and remove proteins from reaction mixture, samples were mixed vigorously and centrifuged at 29900 x g, 4 °C for 15 min.1 µL of supernatant was used for HPLC-MS / MS analysis. We collected three ion transitions to detect N1-methyl-Ψ in the 25 analysed samples: 257.2 [M-H]- → 124.00; 259.2 [M+H]+→ 223.05; and 259.0 [M+H]+→ 205.10. 30 Peaks with retention time of 2.091-2.099 min had the mentioned ion m / zs which closely match that of N1-methyl-Ψ with its exact molecular mass of 258.0852 Da, as well as that of a commercially-obtained standard. Mass chromatograms recorded for a reaction mixture comprising S17 are shown in Fig.14, and analogous chromatograms were observed for the other enzymes investigated in this example. Fig.15 is a mass chromatogram of a control mixture. The control mixture comprised all of the components of the reaction mixture except for the enzyme, and was spiked with 1.44 pmol of a commercial N1-methylpseudouridine 5 standard. Example 8. Enzymatic production of N1-methylpseudouridine coupled with SAM recycling The coupling of pseudouridine methylation by Class I methyltransferases as descrived herein 10 with two SAM recycling systems was investigated. The first SAM recycling system utilizes a methyl halide transferase according to SEQ ID NO: 29 from the acidobacterium Chloracidobacterium thermophilum (HMT) and CH3I methyl donor for SAH methylation (see Liao, C., Seebeck, F.P. (2019). S-adenosylhomocysteine as a methyl 15 transfer catalyst in biocatalytic methylation reactions. Nat Catal 2, 696–701. https: / / doi.org / 10.1038 / s41929-019-0300-0). The second SAM recycling system uses a thiopurine methyltransferase (TPMT) according to SEQ ID NO: 30 from Ustilago maydis (uma) and methyl p-toluenesulfonate (MeOTs) methyl 20 donor for SAH methylation (see Wen, X., Leisinger, F., Leopold, V., & Seebeck, F. P. (2022). Synthetic Reagents for Enzyme-Catalyzed Methylation. Angew.Chem. Int.Ed., 61(41). https: / / doi.org / 10.1002 / anie.202208746). Genes of the recycling system enzymes HMT and uma were synthesized (Twist Bioscience). 25 Plasmids carrying these genes were transformed into E. coli using electroporation: electro- competent E. coli subjected to 18,000 V / cm. A 5'-methylthioadenosine / S- adenosylhomocysteine nucleosidase-deficient strain of E. coli was used to avoid S- adenosylhomocysteine (SAH) degradation. Expression was carried out in media consisting of 2 % tryptone (Formedium), 1 % yeast extract (Formedium), 2 % NaCl (Roth) and 100 µg / mL 30 ampicillin (Sigma-Aldrich) using a T7 RNA polymerase / promoter system. The cells were grown at 37 °C, induced with 0.5 mM IPTG (Sigma-Aldrich) and 0.1 % L-rhamnose (Roth) at OD 600 = 0.8 and grown for 24 h at 16 °C afterwards. The proteins were expressed with 6 histidine residues in the C-terminal (uma) or N-terminal (HMT) to facilitate purification. Reaction mixtures containing 50 mM sodium phosphate buffer, pH 8.0, 4 mM methyl donor (CH3I or MeOTs), 2 mM pseudouridine, 20 μM SAH, 12 μM SAM recycling enzyme (HMT or uma) and 10 μM S17 methyltransferase in a total volume of 20 μl were incubated with shaking in 25 °C for 24h. The identity and yield of the reaction product (synthesized N1-methyl-Ψ) and proof of enzyme specificity was confirmed by HPLC-MS / MS. For HPLC-MS / MS analysis, 3 μl of reaction mixture was diluted with 14 μl of MilliQ water and 34 μl of acetonitrile. To denature and remove proteins, samples were mixed vigorously and centrifuged at 29900 x g, 4 °C for 15 min.1 μl of supernatant was used for HPLC-MS / MS analysis. To quantify the amount of synthesized product N1-methyl-Ψ calibration samples with known N1-methyl-Ψ concentrations were prepared. Number of cycles was determined as: [N1-methyl-Ψ] / [SAH]t=0. As seen in Table 1 below, S17 enzyme coupled with CH3I dependent HMT enzyme synthesized 220.9 μM of product N1-methyl-Ψ, which corresponds to about 11 completed reaction cycles. When coupled with MeOTs dependent enzyme uma, S17 enzyme synthesized 1.12 mM N1- methyl-Ψ, which corresponds to about 56 completed reaction cycles. It should be noted that some N1-methyl-Ψ was detected in the control reaction without enzymes, probably due to spontaneous methylation of Ψ by MeOTs. However, the N1-methyl-Ψ peak area was lower than that of the lowest concentration calibration sample, which means that concentration of product in the reaction is less than 34 μM. Thus, spontaneous methylation of Ψ by MeOTs is negligible compared to the enzymatic methylation. Table 1. Result summary of enzymatic N1-methylpseudouridine synthesis coupled with SAM recycling. n.d. - not detected. Number of SAM N1-methyl-Ψ, μM regeneration cycles CH3I based recycling Reaction mixture with all components 220.9 11 Control without S17 enzyme n.d. - Control without HMT enzyme n.d. - Control without S17 and HMT enzymes n.d. - Control without CH3I n.d. - MeOTs based recycling Reaction mixture with all components 1119.6 56 Control without S17 enzyme < 34 - Control without uma enzyme < 34 - Control without S17 and uma enzymes < 34 - Control without MeOTs n.d. - Example 9. Enzymatic production of N1-methyl pseudouridine in vivo E. coli cells were transformed by recombinant plasmids harbouring genes of enzymes S17 and 5 S19 or an empty control vector (negative control) by electroporation. Expression was carried out in media comprising 2 % tryptone (Formedium), 1 % yeast extract (Formedium), 2 % NaCl (Roth) and 100 µg / mL ampicillin (Sigma-Aldrich) using a T7 RNA polymerase / promoter system. The cells were grown at 37 °C, induced with 0.5 mM IPTG (Sigma-Aldrich) and 0.1 % L-rhamnose (Roth) at OD 600 = 0.8 and grown for 20 h at 16 °C. 10 Afterwards, 1.5 mL of each cell culture was collected by centrifugation, washed with 1 mL of 50 mM potassium phosphate buffer (pH 7.5) and resuspended into 100 μL of the same buffer that contained 5 mM of pseudouridine (Ψ). Tubes containing reaction mixtures were incubated at 37 °C and 180 rpm for 3 days. The cells were separated from the buffer by 15 centrifugation. The identity and yield of reaction product (synthesized N1-methyl-Ψ) and proof of enzyme specificity was confirmed by HPLC-MS / MS. The supernatant was mixed with acetonitrile (1:1), centrifuged at 29900 x g, 4 °C for 15 min. We collected three ion transitions to detect N1- methyl-Ψ in the analysed samples: 257.2 [M-H]- → 124.00, 259.2 [M+H]+→ 223.05 and 259.0 [M+H]+→ 205.10. Peaks with retention time of 2.091-2.099 min had the mentioned ion m / zs which closely match that of N1-methyl-Ψ with its exact molecular mass of 258.0852 Da. N1- methyl pseudouridine concentration was determined according to a calibration curve 5 determined using a standard. As shown in Table 2, N1-methyl pseudouridine can be produced in vivo using a microorganism that expresses a Class I methyltransferase as provided herein. 10 Table 2. Enzymatic N1-methyl pseudouridine production in vivo result summary N1-methyl-Ψ yield, % N1-methyl-Ψ, μM S17 buffer 18.02 901.1 S19 buffer 9.3 464.9 Control buffer 0 0 It will be appreciated that the above embodiments have been described by way of example only. 15 Other variants or use cases of the disclosed techniques may become apparent to the person skilled in the art once given the disclosure herein. The scope of the disclosure is not limited by the described embodiments but only by the accompanying claims.

Claims

Claims 1. A method of synthesising a 1N-alkyl derivative of pseudouridine, which method comprises: contacting a substrate with a methyltransferase in the presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine; wherein: the substrate has a structure of:the 1N-methyl derivative of pseudouridine has a structure of:R1being a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group; R2being H or a hydroxyl group; and R3being a methyl group or an ethyl group; the cofactor is a source of a methyl group or an ethyl group; and the methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate.

2. The method according to claim 1, wherein producing the 1N-alkyl derivative of pseudouridine further produces a dealkylated cofactor, and wherein the method further comprises regenerating the cofactor by alkylating the dealkylated cofactor.

3. The method according to claim 2, wherein the cofactor is regenerated using an alkyl group donor in the presence of a catalyst.

4. The method according to claim 3, wherein the alkyl group donor is an alkyl halide and the catalyst is a methyl halide transferase, optionally wherein the cofactor is S-adenosyl-L-methionine, the alkyl halide is methyl iodide, and the methyl halide transferase is a methyl halide transferase from Chloracidobacterium thermophilum (SEQ ID NO:29).

5. The method according to claim 3, wherein the alkyl group donor is an alkyl toluenesulfonate and the catalyst is a thiopurine alkyltransferase; optionally wherein the alkyl group donor is methyl p-toluenesulfonate and the thiopurine alkyltransferase is a thiopurine methyltransferase from Ustilago maydis (SEQ ID NO:30).

6. The method according to any preceding claim, wherein the cofactor is S-adenosyl-L- methionine or S-adenosyl-L-ethionine.

7. The method according to claim 6, wherein R3is a methyl group and the cofactor is S- adenosyl-L-methionine.

8. The method according to any preceding claim, wherein R1is a hydroxyl group, a monophosphate group, or a triphosphate group.

9. The method according to claim 8, wherein R1is a hydroxyl group or a monophosphate group, and the method further comprises, after the contacting, phosphorylating the 1N-alkyl derivative of pseudouridine.

10. The method according to any preceding claim, wherein R2is a hydroxyl group.

11. The method according to any preceding claim, further comprising preparing the substrate by contacting D-ribose-5-monophosphate with uracil and a pseudouridine monophosphate glycosidase to form pseudouridine 5'-monophosphate.

12. The method according to claim 11, further comprising contacting the pseudouridine 5'-monophosphate with a phosphatase to form pseudouridine.

13. The method according to claim 11 or claim 12, further comprising forming the D- ribose-5-monophosphate by contacting α-D-ribose-1-monophosphate with a phosphopentomutase.

14. The method according to claim 13, further comprising forming the D-ribose-5- monophosphate by contacting uridine with a uridine phosphorylase and an inorganic phosphate.

15. The method according to any preceding claim, further comprising producing the methyltransferase by fermentation of a microorganism configured to express the methyltransferase; optionally wherein the microorganism is further configured to express uridine phosphorylase, phosphopentomutase, and pseudouridine monophosphate glycosidase.

16. The method according to claim 15, wherein the contacting is performed in the presence of the microorganism; optionally wherein the method is performed in vivo.

17. The method according to any preceding claim, wherein the contacting is performed in an aqueous buffer, the aqueous buffer including Mg2+at a concentration in the range 0.5 mM to 15 mM, optionally 0.7 mM to 1.3 mM, further optionally 0.9 to 1.1 mM.

18. The method according to claim 17, wherein the buffer has a pH in the range 7.4 to 7.6.

19. The method according to any preceding claim, wherein the methyltransferase has an active site with a neutral charge or a positive charge, optionally wherein the active site includes a lysine or arginine residue positioned to interact with atom O4 of the substrate and / or the active site has a net positive charge of one or two.

20. The method according to any preceding claim, wherein the methyltransferase is a Class I methyltransferase engineered to transfer the methyl group or ethyl group from the cofactor to the substrate.

21. The method according to claim 20, wherein the methyltransferase is a xanthosine methyltransferase.

22. The method according to claim 21, wherein the methyltransferase has an amino acid sequence which includes a portion having at least 90 % sequence identity to residues 1 to 306 of SEQ ID NO:

19.

23. The method according to claim 22, wherein the methyltransferase has an amino acid sequence with at least 90 % sequence identity to SEQ ID NO: 19, the amino acid sequence including one or more mutations selected to increase stability and / or activity compared with the methyltransferase of SEQ ID NO:

19.

24. The method according to claim 23, wherein the one or more mutations are: i) at least one mutation selected from Q161H and A238S; and ii) optionally one or more mutations selected from: R8H, A23S, V40I, K86M, K86E, I97V, P104Q, G135S, G135A, S143G, C157S, C159S, S174L, T178A, R190P, L191P, H212R, F217L, H219R, V231D, L233S, A235G, R236P, L265V, P270A, L296R, D304_D305Ins(DDYQVRSHSPVYC), I308A, K309R, E311A, Y312H, A319S ,I337L, H344N, and P350R.

25. The method according to claim 24, wherein the methyltransferase has a sequence selected from: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO:

27.

26. The method according to claim 22, wherein the methyltransferase has an amino acid sequence with at least 90 % sequence identity to SEQ ID NO:

20.

27. The method according to claim 26, wherein the methyltransferase has an amino acid sequence according to SEQ ID NO:

28.

28. The method according to claim 20, wherein the methyltransferase is a cap methyltransferase, optionally an mRNA cap guanine-N7 methyltransferase.

29. The method according to claim 28, wherein the methyltransferase has an amino acid sequence with at least 80 % sequence identity to SEQ ID NO: 5, the amino acid sequence including at least one mutation selected from: i) F201R or F201K; ii) E212Q, E212K, E212R, E212N, E212S, E212C, E212A, E212T, E212M, E212H, E212G, E212V, E212L, or E212I; iii) I267R or I267K; iv) Y271R or Y271K; optionally wherein the amino acid sequence includes no more than two mutations selected from i) to iv).

30. The method according to claim 28, wherein the methyltransferase has an amino acid sequence with at least 80 % sequence identity to SEQ ID NO: 6, the amino acid sequence including at least one mutation selected from: i) F215R or F215K; ii) E226Q, E226K, E226R, E226N, E226S, E226C, E226A, E226T, E226M, E226H, E226G, E226V, E226L, or E226I; iii) A286R or A286K; iv) Y290R or Y290K;optionally wherein the amino acid sequence includes no more than two mutations selected from i) to iv).

31. The method according to claim 28, wherein the methyltransferase has an amino acid sequence with at least 80 % sequence identity to SEQ ID NO: 7, the amino acid sequence including at least one mutation selected from: i) Y224R or Y224K; ii) E235Q, E235K, E235R, E235N, E235S, E235C, E235A, E235T, E235M, E235H, E235G, E235V, E235L, or E235I; iii) A299R or A299K; iv) Y304R or Y304K; optionally wherein the amino acid sequence includes no more than two mutations selected from i) to iv).

32. The method according to claim 28, wherein the methyltransferase has an amino acid sequence with at least 80 % sequence identity to SEQ ID NO: 8, the amino acid sequence including at least one mutation selected from: i) F199R or F199K; ii) E210Q, E210K, E210R, E210N, E210S, E210C, E210A, E210T, E210M, E210H, E210G, E210V, E210L or E210I; iii) V265R or V265K; and iv) Y269R or Y269K; optionally wherein the amino acid sequence includes no more than two mutations selected from i) to iv).

33. The method according to any of claims 1 to 19, wherein the methyltransferase is a dimeric SPOUT methyltransferase comprising a SPOUT domain configured to transfer the methyl group from the cofactor to the substrate.

34. The method according to claim 33, wherein the methyltransferase is a minimalist dimeric SPOUT methyltransferase, free of any domains other than the SPOUT domain.

35. The method according to claim 34, wherein the methyltransferase consists of the SPOUT domain.

36. The method according to any preceding claim, wherein the methyltransferase has an active site which includes an amino acid residue which forms a stacking interaction with the substrate when the SPOUT domain binds to the substrate, optionally wherein the amino acid residue which forms the stacking interaction is arranged proximal to one or more amino acid residues which catalyse transfer of the methyl group or ethyl group from the cofactor to the substrate.

37. The method according to claim 36, wherein the amino acid residue which forms the stacking interaction is selected from a histidine residue, a tyrosine residue, and a tryptophan residue; optionally wherein the amino acid residue which forms the stacking interaction is a tyrosine residue.

38. The method according to any preceding claim, wherein R1is a monophosphate group, a diphosphate group, or a triphosphate group, wherein the methyltransferase includes an amino acid residue which forms a salt bridge with a phosphate group of the substrate when the methyltransferase binds to the substrate; and optionally wherein the amino acid residue which forms the salt bridge is a lysine or arginine residue.

39. The method according to any preceding claim, wherein the methyltransferase has a neutrally-charged or positively-charged active site.

40. The method according to any of claims 33 to 39, wherein the methyltransferase is a tRNA (pseudouridine54-N1)-methyltransferase.

41. The method according to claim 40, wherein the methyltransferase is an engineered tRNA (pseudouridine54-N1)-methyltransferase which includes at least one of the following modifications compared to wild-type tRNA (pseudouridine54-N1)-methyltransferase: i) introduction of a stacking amino acid residue selected from a histidine residue, a tyrosine residue, and a tryptophan residue, the stacking amino acid residue being arranged proximal to one or more amino acid residues which catalyse transfer of the methyl group or ethyl group from the cofactor to the substrate; and / or ii) introduction of a salt bridging amino acid residue configured to form a salt bridge to a phosphate group of the substrate, the salt bridging amino acid being selected from a lysine residue and an arginine residue, and the salt bridging amino acid being arranged proximal to one or more amino acid residues which catalyse transfer of the methyl group or ethyl group from the cofactor to the substrate; and / or iii) replacement of a negatively-charged amino acid residue proximal to one or more amino acid residues which catalyse transfer of the methyl group or ethyl group from the cofactor to the substrate with a neutrally-charged or positively-charged amino acid residue, preferably a positively-charged amino acid residue.

42. The method according to claim 41, wherein the methyltransferase has an amino acid sequence having at least 70 % sequence identity to SEQ ID NO:

1.

43. The method according to claim 42, wherein the amino acid sequence includes mutations K10Q, K75N, and one of K20D and K20N.

44. The method according to claim 42 or claim 43, wherein the wherein the amino acid sequence includes one or more mutations selected from K74D, R81L, and K89N.

45. The method according to any of claims 42 to 44, wherein the amino acid sequence includes one or more mutations selected from: D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, I193V, K199R, K200R, R201E, and C203F.

46. The method according to any of claims 42 to 45, wherein the amino acid sequence includes one or more mutations selected from: D104Y, R115K, N121D, V123I, M138K, N139D, I155L, R167K, R176K, K200R, R201N, C203del, E204del, and I205del.

47. The method according to any of claims 42 to 46, wherein the amino acid sequence includes one or more mutations selected from: L32V, D38S, Q59E, C65A, S71N, E100N, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, I154V, I155L, R167K, D170E, K173G, R176K, I177V, N187D, I193V, K200R, R201G, C203del, E204del, and I205del.

48. The method according to any of claims 42 to 47, wherein the amino acid sequence includes a P23K mutation.

49. The method according to claim 48, wherein the amino acid sequence further includes an S77D mutation.

50. The method according to any of claims 42 to 49, wherein the amino acid sequence includes an F41N mutation.

51. The method according to any of claims 42 to 50, wherein the amino acid sequence includes one or more mutations selected from K10Q, P23D, S77K, and D79N.

52. The method of claim 42, wherein the methyltransferase has an amino acid sequence selected from: i) SEQ ID NO: 1 with mutations K10Q, K20D, and K75N; ii) SEQ ID NO: 1 with mutations K10Q, K20N, K75N, and R81L; iii) SEQ ID NO: 1 with mutations K10Q, K20D, K75N, K74D, and K89N; iv) SEQ ID NO: 1 with mutations D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, I193V, K199R, K200R, R201E, and C203F; v) SEQ ID NO: 1 with mutations K10Q, P23D, S77K, and D79N;vi) SEQ ID NO: 1 with mutations D104Y, R115K, N121D, V123I, M138K, N139D, I155L, R167K, R176K, K200R, R201N, C203del, E204del,and I205del; and vii) SEQ ID NO: 1 with mutations L32V, D38S, Q59E, C65A, S71N, E100N, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, I154V, I155L, R167K, D170E, K173G, R176K, I177V, N187D, I193V, K200R, R201G, C203del, E204del, and I205del.

53. The method according to claim 40 or claim 41, wherein the methyltransferase has an amino acid sequence having at least 70 % sequence identity to SEQ ID NO:

2.

54. The method according to any of claims 33 to 39, wherein the methyltransferase is an rRNA small subunit pseudouridine methyltransferase Nep1.

55. The method according to claim 54, wherein the methyltransferase is an engineered methyltransferase having: i) a stacking amino acid residue selected from a histidine residue, a tyrosine residue, and a tryptophan residue, the stacking amino acid residue being arranged proximal to one or more amino acid residues which catalyse transfer of the methyl group or ethyl group from the cofactor to the substrate; and ii) a neutrally-charged or positively charged active site for catalysing transfer of the methyl group or ethyl group from the cofactor to the substrate.

56. The method according to claim 54 or claim 55, wherein the methyltransferase has an amino acid sequence having 70 to 99.6 % sequence identity to SEQ ID NO:3, the amino acid sequence including one or more mutations to SEQ ID NO:

3.

57. The method according to claim 56, wherein the at least one mutation comprises V2_A27del.

58. The method according to claim 56 or claim 57, wherein the one or more mutations comprise at least one of K106Q, R129N, and S233W.

59. The method according to claim 58, wherein the one or more mutations comprise S223W and one or more of L97A, D101A, and R132S; preferably wherein the mutations are S223W, L97A, D101A, and R132S.

60. The method according to claim 54 or claim 55, wherein the methyltransferase has an amino acid sequence having 70 to 99.6 % sequence identity to SEQ ID NO: 4, the amino acid including one or more mutations.

61. The method according to claim 60, wherein the one or more mutations comprise D210H, D210Y, D210P, or D210W; and optionally one or more of: L131, V132L, L133V, M134L, Y135M, V136Y, K137V, Q139K, S140Q, L141S, E142, G143L, L144E, L145G, K146L, E147L, I148K, K149E, P150I, T151K, R152P, F154R, M155T, L156F, S157M, E158L, N159S, G160E, E161N, K162G, I163E, N164K, S166N, T167I, L168S, A169T, K170L, E171A, L172K, C173E, Q174L, E175C, N176Q, R177E, P178N, T179R, V180P, I181T, I182V, G183I, G184I, F185G, Q186G, K187F, G188Q, S189H, F190G, L191S, K192F, K193L, H194K, V195K, E196H, L197V, A198E, D199L, K200A, V201D, Y202K, A203V, V204Y, Y205A, G206V, S207Y, P208G, L209S, D210P, T211L, V213T, I214A, A215V, S216I, M217A, L218S, L219M, H220L, G221L, Y222H, E223G, I224Y, K226I, G227E, I228K, I229G.

62. The method according to claim 61, wherein the mutations further comprise R107S.

63. The method according to claim 61 or claim 62, wherein the mutations further comprise W212A, W212H, or W212N.

64. The method according to claim 63, wherein the mutations comprise: a) D210H and W212H; or b) D210Y and W212N.

65. The method according to any of claims 61 to 64, wherein the mutations further comprise I45K.

66. The method according to claim 65, wherein the mutations further comprise Y49E.

67. The method according to any of claims 61 to 66, wherein the mutations further comprise G76A or G76N.

68. The method according to any of claims 61 to 67, wherein the mutations comprise D210Y and Q186A.

69. The method according to any of claims 60 to 68, wherein the mutations comprise one or more of: K104N, R111Q, and K187H.

70. The method according to claim 64, wherein the mutations are I45K, Y49E, G76A, K104N, R107S, R111Q, Q186A, K187H, D210Y, and W212N.

71. A methyltransferase having an amino acid sequence of SEQ ID NO: 1 with one or more mutations, the one or more mutations being selected from: R116K, V143I, S77K, N106T, R201G, R81L, R115K, S71N, M138K, K20D, K199R, I205del, Q101K, I154V, E128K, N139D, V123I, L117K, E204del, N121D, R201E, D104Y, C203F, L32V, I177V, D147K, I66L, D38S, D104E, N132K, P151_V152insN, N150_P151insE, D79N, K173G, Q107E, K200R, R201N, E149K, R115S, N187D, E149_N150insN, K20N, P23D, R176K, K10Q, N145E, K89N, I155L, I193V, Q59E, E100N, K103E, K75N, L127K, D170E, C65A, C203del, K74D, and R167K.

72. The methyltransferase according to claim 71, wherein the mutations are: i) K10Q, K20D, and K75N; or ii) K10Q, K20N, K75N, and R81L; or iii) K10Q, K20D, K75N, K74D, and K89N; or iv) D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, I193V, K199R, K200R, R201E, and C203F; or v) K10Q, P23D, S77K, and D79N; orvi) D104Y, R115K, N121D, V123I, M138K, N139D, I155L, R167K, R176K, K200R, R201N, C203del, E204del, and I205del; or vii) L32V, D38S, Q59E, C65A, S71N, E100N, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, I154V, I155L, R167K, D170E, K173G, R176K, I177V, N187D, I193V, K200R, R201G, C203del, E204del, and I205del.

73. A methyltransferase having an amino acid sequence of SEQ ID NO: 3 with a S233W mutation and optionally one or more further mutations selected from V2_A27del, L97A, D101A, K106Q, R129N and R132S.

74. The methyltransferase according to claim 62, wherein the further mutations are L97A, D101A, and R132S.

75. A methyltransferase having an amino acid sequence of SEQ ID NO: 4 with a mutation selected from D210H, D210Y, D210P, and D210W; and optionally one or more further mutations selected from I45K, Y49E, G76A, G76N, K104N, R107S, R111Q, Q186A, K187H, W212A, W212H, W212N, L131, V132L, L133V, M134L, Y135M, V136Y, K137V, Q139K, S140Q, L141S, E142, G143L, L144E, L145G, K146L, E147L, I148K, K149E, P150I, T151K, R152P, F154R, M155T, L156F, S157M, E158L, N159S, G160E, E161N, K162G, I163E, N164K, S166N, T167I, L168S, A169T, K170L, E171A, L172K, C173E, Q174L, E175C, N176Q, R177E, P178N, T179R, V180P, I181T, I182V, G183I, G184I, F185G, Q186G, K187F, G188Q, S189H, F190G, L191S, K192F, K193L, H194K, V195K, E196H, L197V, A198E, D199L, K200A, V201D, Y202K, A203V, V204Y, Y205A, G206V, S207Y, P208G, L209S, D210P, T211L, V213T, I214A, A215V, S216I, M217A, L218S, L219M, H220L, G221L, Y222H, E223G, I224Y, K226I, G227E, I228K, and I229G; or one or more further mutations selected from: I45K; Y49E; G76A or G76N; K104N; R107S; R111Q; K187H; Q186A; and W212A, W212H, or W212N.

76. The methyltransferase according to claim 75, wherein the mutations comprise:a) D210H and W212H; or b) D210Y and W212N.

77. The methyltransferase according to claim 75 or claim 76, wherein the mutations include I45K and Y49E.

78. The methyltransferase according to any of claims 75 to 77, wherein the mutations include G76A or G76N.

79. The methyltransferase according to claim 75, wherein the mutations are D210Y and Q186A.

80. The methyltransferase according to claim 75, wherein the mutations are I45K, Y49E, G76A, K104N, R107S, R111Q, Q186A, K187H, D210Y, and W212N.

81. A methyltransferase having an amino acid sequence of SEQ ID NO: 18 with at least one of the following mutations: I45K, Y49E, G76A, K104N, R107S, R111Q, K187H, V201, Y202V, A203Y, V204A, Y205V, G206Y, S207G, P208S, L209P, D210L, T211H, W212, V213T, I214A, A215V, S216I, M217A, L218S, L219M, H220L, G221L, Y222H, E223, I224G, E225Y, K226E, G227I, I228E, and I229K.

82. The methyltransferase according to claim 81, having an amino acid sequence of SEQ ID NO:

12.

83. A methyltransferase having an amino acid sequence of SEQ ID NO: 5 with at least one mutation selected from: i) F201R or F201K; ii) E212Q, E212K, E212R, E212N, E212S, E212C, E212A, E212T, E212M, E212H, E212G, E212V, E212L, or E212I; iii) I267R or I267K; iv) Y271R or Y271K.

84. A methyltransferase having an amino acid sequence of SEQ ID NO: 6 with at least one mutation selected from: i) F215R or F215K; ii) E226Q, E226K, E226R, E226N, E226S, E226C, E226A, E226T, E226M, E226H, E226G, E226V, E226L, or E226I; iii) A286R or A286K; iv) Y290R or Y290K.

85. A methyltransferase having an amino acid sequence of SEQ ID NO: 7 with at least one mutation selected from: i) Y224R or Y224K; ii) E235Q, E235K, E235R, E235N, E235S, E235C, E235A, E235T, E235M, E235H, E235G, E235V, E235L, or E235I; iii) A299R or A299K; iv) Y304R or Y304K.

86. A methyltransferase having an amino acid sequence of SEQ ID NO: 8 with at least one mutation selected from: i) F199R or F199K; ii) E210Q, E210K, E210R, E210N, E210S, E210C, E210A, E210T, E210M, E210H, E210G, E210V, E210L or E210I; iii) V265R or V265K; iv) Y269R or Y269K.

87. The methyltransferase of any of claims 83 to 86, wherein the amino acid sequence includes no more than two mutations.

88. A methyltransferase having an amino acid sequence which includes a portion having at least 90 % sequence identity to residues 1 to 306 of SEQ ID NO: 19, and which includes at least one mutation selected from Q161H and A238S.

89. The methyltransferase according to claim 88, wherein the methyltransferase has an amino acid sequence with at least 90 % sequence identity to SEQ ID NO:

19.

90. The methyltransferase according to claim 89, having one or more mutations selected from: R8H, A23S, V40I, K86M, K86E, I97V, P104Q, G135S, G135A, S143G, C157S, C159S, S174L, T178A, R190P, L191P, H212R, F217L, H219R, V231D, L233S, A235G, R236P, L265V, P270A, L296R, D304_D305Ins(DDYQVRSHSPVYC), I308A, K309R, E311A, Y312H, A319S ,I337L, H344N, and P350R.

91. The methyltransferase according to claim 88, having an amino acid sequence selected from SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO:

27.

92. The method according to claim 88, wherein the methyltransferase has an amino acid sequence with at least 90 % sequence identity to SEQ ID NO:

20.

93. The method according to claim 92, wherein the methyltransferase has an amino acid sequence according to SEQ ID NO:

28.

94. A polynucleotide having a sequence encoding a methyltransferase as defined in any of claims 71 to 93.

95. An expression vector carrying the polynucleotide of claim 94.

96. A microorganism transduced with the expression vector of claim 95.

97. Use of an aqueous buffer as a reaction medium for a method of synthesising a 1N- alkyl derivative of pseudouridine as defined in any of claims 1 to 70, wherein the aqueous buffer includes the substrate, the cofactor, the methyltransferase, and Mg2+, the Mg2+being present at a concentration in the range 0.5 mM to 15 mM; and the aqueous buffer has a pH in the range 7.4 to 7.6.

98. Use according to claim 97, wherein the aqueous buffer is a Tris buffer.

99. Use according to claim 97 or claim 98, wherein the aqueous buffer includes NaCl at a concentration of 50 to 350 mM, and KCl at a concentration of 70 to 130 mM.

100. Use according to any of claims 97 to 99, wherein the Mg2+is supplied by MgCl2.

101. Use according to any of claims 97 to 100, wherein: the cofactor is present in the aqueous buffer at a concentration in the range 20 to 30 µM; and / or the substrate is present in the aqueous buffer at a concentration in the range 80 to 120 µM; and / or the methyltransferase is present in the aqueous buffer at a concentration in the range 0.5 to 20 µM.

102. Use according to claim 101, wherein the buffer has a pH of 7.5 ± 0.1 and includes: Tris-HCl at a concentration of about 25 mM; NaCl at a concentration of about 300 mM; MgCl2at a concentration of about 1 mM; KCl at a concentration of about 100 mM; the cofactor at a concentration of about 25 µM; and the substrate at a concentration of about 100 µM.

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