Synthesis of 1N-methylpseudouridine and 1N-methylpseudouridine phosphates

Abstract

A method of synthesising a IN-alkyl derivative of pseudouridine comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the I N-alkylderivative of pseudourid

Description

lN-methylpseudouridine (lN-methyl-tP) nucleoside and its various phosphorylated derivatives (such as lN-methylpseudouridine-5'-monophosphate (IN-methyl-tPMP) and 1N-methylpseudouridine-5'-triphosphate (lN-methyl-UJTP)) 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, Nl-methylpseudouridine is used instead of uridine in 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 lN-methylpseudouridine nucleoside and its derivatives. One existing process for synthesising lN-methylpseudouridine and its derivatives is illustrated in Fig. 1. Pseudouridine 101 is obtained from natural sources. Chemical methylation is performed to obtain IN-methylpseudouridine 102. Chemical methylation involves protection and deprotection steps in addition to the use of a methylating agent such as methyl iodide. IN-methylpseudouridine monophosphate 103 and lN-methylpseudouridine triphosphate 104 are obtainable by chemical phosphorylation of the lN-methylpseudouridine 102. An alternative existing process involves extracting lN-methylpseudouridine from natural sources. 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 lN-alkyl derivative of pseudouridine. The method comprises contacting a substrate with a methyltransferase in the 5 presence of a cofactor to produce the lN-a Iky I derivative of pseudouridine. The substrate has a structure of: and the lN-methyl derivative of pseudouridine has a structure of: In the formulae above, R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group; R2 is H or a hydroxyl group; and R3 is 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. Still another aspect provides the use of an aqueous buffer as a reaction medium for the synthesis of the IN-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. 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. 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 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" 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) 4>, (b) MJMP, (c) IPTP, and (d) no substrate. 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. Fig. 5 (a comparative example) shows a mass chromatogram of commercially-available lN-methylpseudouridine standard. Figs 6A to 6C are a series of mass chromatograms that confirm that the present method produced lN-methylpseudouridine. The chromatograms are of a reaction mixture containing enzyme N1 and pseudouridine-5'-monophosphate. Fig. 6A is a mass chromatogram of the reaction mixture alone. Fig. 6B is a mass chromatogram of the reaction mixture spiked with 5 picomoles of lN-methylpseudouridine standard. Fig. 6C is an overlayed view of the unspiked reaction mixture and the same reaction mixture spiked with 5 picomoles of lN-methylpseudouridine standard. Fig. 7 is a pairwise identity matrix of natural (N) and engineered (E) SPOUT 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 Nl, E5 and E8, as described in Example 3. Fig. 9A shows the concentration of SAH detected when ethylation of ip and tPMP was conducted using enzyme E5, as discussed in Example 4. Fig. 9B shows the concentration of SAH detected when ethylation of 4>MP was conducted using enzyme E6, as discussed in Example 4. Fig. 10 is a pairwise identity matrix for four mRNA cap guanine-N7 methyltransferases SI to S4. Fig. 11 is a reaction scheme outlining an example method of synthesising lN-methyl psuedouridine from uridine. Detailed Description General definitions 5 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. 10 The symbol "UJ" refers to pseudouridine. "<PI\ / IP" refers to pseudouridine monophosphate. "iPTP" refers to pseudouridine triphosphate. WTP "dip" refers to deoxypseudouridine. "diPMP" refers to deoxypseudouridine monophosphate. 15 "dUJTP" refers to deoxypseudouridine triphosphate. "SAM" stands forS-adenosyl-L-methionine: SAM "SAH" stands forS-adenosylhomocysteine. As used herein, the expression "lN-alkyl derivative of pseudouridine" refers to a compound of Formula 2: Formula 2 where: R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group; R2 is H or a hydroxyl group; and R3 is a methyl group or an ethyl group. The methods provided herein most typically produce lN-methyl derivatives of pseudouridine, i.e. compounds of Formula 2 in which R3 is a methyl group. "Phosphorylated" compounds are compounds in which R1 is 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. 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. Methods of synthesising lN-alkyl derivatives of pseudouridine Provided herein is a method of synthesising a lN-alkyl derivative of pseudouridine. The method comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the lN-alkyl derivative of pseudouridine. The method may be performed in vivo or ex vivo. The substrate has a structure of Formula 1: Formula 1 R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group, and R2 is H or OH. In other words, the substrate is an optionally-phosphorylated pseudouridine or deoxypseudouridine. Atom 04 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 04 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 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. 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 a-D-ribose-l-monophosphate; ii) contacting the a-D-ribose-l-phosphate with a pentomutase to obtain D-ribose-5-monophosphate; 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. Steps i) to iii) and optionally iv) may be performed in vivo, particularly in implementations where the synthesis of the lN-alkyl derivative of pseudouridine is performed in vivo. The lN-alkyl derivative of pseudouridine produced by the method has a structure of Formula 2: Formula 2 Contacting the substrate with the methyltransferase does not modify R1 or R2. R3 is 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 transferring the methyl group or ethyl group from the cofactor to the substrate to produce the lN-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 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 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 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 04 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 R1 is 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 orS-adenosyl-L-ethionine. Most typically, the methods provided herein produce a IN-methyl derivative of pseudouridine (i.e., R3 is a methyl group). The cofactor is most typically S-adenosyl-L-methionine. Optionally, R1 is selected from a hydroxyl group, a monophosphate group, and a triphosphate group. When R1 is a hydroxyl group or a monophosphate group, the method may further comprise, after the contacting, phosphorylating the lN-alkyl derivative of pseudouridine. The phosphorylation may be chemical phosphorylation. R2 may be a hydroxyl group. In other words, the substrate may be an optionally-phosphorylated pseudouridine. Alternatively, R2 may 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 lN-alkyl derivative of pseudouridine may take place in vivo. In such implementations, the lN-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 MgCb. 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: NaCI at a concentration of 50 to 350 mM, optionally 270 to 230 mM, further optionally 290 to 310 mM; and / or KCI 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 present at a concentration in the range 20 to 30 pM, and the substrate may be present at a concentration in the range 80 to 120 pM. The concentration of the methyltransferase is typically in the range 0.5 to 20 pM. In particular, the buffer may have a pH of 7.5 ± 0.1 and include: Tris-HCI at a concentration of about 25 mM; NaCI at a concentration of about 300 mM; MgCl2 at a concentration of about 1 mM; KCI at a concentration of about 100 mM; the cofactor (e.g., SAM) at a concentration of about 25 pM; and the substrate at a concentration of about 100 pM. One aspect provides a use of the buffers discussed above as a reaction medium for the enzymatic synthesis by a methyltransferase of a lN-alkyl derivative of pseudouridine according to Formula 2 from a substrate of Formula 1 and a cofactor selected from S-adenosyl-L-methionine and S-adenosyl-L-ethionine. Example Class I Methyltransferases 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 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. 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). 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. 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 preferably, the positive charge is positioned to interact with atom 04 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. 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, E212V, E212L, and E212I; optionally E212K or E212R, and preferably E212R; ii) F201R or F201K; iii) I267R or I267K; iv) Y271R or Y271K. 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). 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; ii) F215Ror F215K; iii) A286R or A286K; iv) Y290R orY290K. Typically, the mutations introduce at least one but no more than two positively-charged 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 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 orY304K. 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 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) F199Ror F199K; 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 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). 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. 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 R1 of 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. Although the above methyltransferases have been described in the context of the method of synthesising a IN-alkyl derivative of pseudouridine, it should be appreciated that the methyltransferases as such represent an independent aspect of the present disclosure. 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 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. 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. 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 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 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 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 techniques. Alternatively or additionally, when group R1 of 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 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-Nl)-methyltransferase. Two examples of tRNA (pseudouridine54-Nl)-methyltransferases were investigated, and both 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-Nl)-methyltransferase has an amino acid sequence of SEQ ID NO: 1. Variants of this methyltransferase which include at least one 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. 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, 1193V, K199R, K200R, R201E, C203F. 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, l205del. Variants of SEQ ID NO: 1 may alternatively or additionally include one or more mutations selected from: L32V, D38S, Q59E, C65A, S71N, EICON, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, 1154V, I155L, R167K, D170E, K173G, R176K, 1177V, N187D, 1193V, K200R, R201G, C203del, E204del, and l205del. 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. Specific examples of useful methyltransferases include the following: i) SEQ ID NO: 1 with mutations K10Q, K20D, and K75N (i.e., Enzyme El 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; iv) SEQ ID NO: 1 with mutations D38S, C65A, I66L, Q107E, N145E, E149K, 1155L, R176K, 1193V, 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 l205del (Enzyme E12, SEQ ID NO: 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, 1154V, I155L, R167K, D170E, K173G, R176K, 1177V, N187D, 1193V, K200R, R201G, C203del, E204del, and 1205del (Enzyme E7, SEQ ID NO: 14). Another example of a useful tRNA (pseudouridine54-Nl)-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. 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. The methyltransferase may alternatively be an rRNA small subunit pseudouridine methyltransferase Nepl engineered to methylate the substrate. The engineered rRNA small subunit pseudouridine methyltransferase Nepl may comprise a mutation compared to a wild-type Nepl, the mutation introducing: 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 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. Alternatively or additionally, the engineered rRNA small subunit pseudouridine methyltransferase Nepl 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 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. Preferably, at least one but no more than two positively-charged residues are introduced at the active site. The rRNA small subunit pseudouridine methyltransferase Nepl may have an amino acid sequence having 70 to 99.6 % sequence identity to SEQ ID NO:3. The amino acid sequence 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 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. 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. 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: 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 Nepl useful in the methods described herein is a methyltransferase having an amino acid sequence having 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 comprise R107S, particularly in implementations which include D210H. Alternatively or additionally, the at least one mutation may comprise W212A, W212H, or W212N. 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. 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. 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, KWH, D210Y, and W212N. 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. Although the above methyltransferases have been described in the context of the method of synthesising a IN-alkyl derivative of pseudouridine, it should be appreciated that the methyltransferases as such represent an independent aspect of the present disclosure. 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 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'-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 a-D-ribose-l-monophosphate with a phosphopentomutase. The a-D-ribose-l-monophosphate may be formed by contacting uridine with a uridine phosphorylase and an inorganic phosphate. The substrate may conveniently be prepared in vivo. Conveniently, both the preparation of the substrate and the synthesis of the IN-a Iky I 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, and the methyltransferase. Pseudouridine-5' -monophosphate may in particular be prepared by the method described in WO 2023 / 131727 Al. Examples Example 1. SPOUT methyltransferases Enzymes potentially capable of methylating pseudouridine substrates were identified by 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. 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. 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 % NaCI (Roth) and 100 pg / mL ampicillin (Sigma-Aldrich) using 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 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 MJ, pseudourdine monophosphate iPMP, and pseudouridine triphosphate OTP. The enzymes tested are discussed further below. Optimised buffer conditions for enzymatic production of Nl-methylpseudouridine and its derivatives were identified by performing reactions in various 25 mM Tris-HCI solutions having different pHs, NaCI concentrations, and MgCI? concentrations. Each solution included 100 mM KCI, 25 pM S-adenosyl-L-methionine (SAM), 100 pM pseudouridine-5'-monophosphate or pseudouridine-5'-triphosphate as a substrate, and 0.7 to 9 pM methyltransferase in a total volume of 25 pL (Table 1). Reaction mixtures were incubated at 37 °C for 24 h and monitored by detecting S-adenosylhomocysteine (SAH), a secondary 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 [NaCI] / mM [MgCh] / mM [KCI] / mM [SAM] / pM Substrate / pM 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-HCI at pH 7.5,100 mM KCI, 300 mM NaCI, ImM MgCl2 gave the best performance. These conditions with slight modifications were used in further experiments. Methylation activity was tested in a reaction mixture containing 25 mM Tris-HCI (pH 7.5), 100 mM KCI, 300 mM NaCI, ImM MgCI2, 0.03 % NP-40, 25 pM 5-adenosyl-L-methionine (SAM), 1 mM of substrate (>P, iPMP or >PTP) and 2.5 to 20 pM methyltransferase (depending on the protein used), in a total volume of 25 pL. 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 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 >P, >PMP, and >PTP as substrates. Only a trace amount of SAH was detected in the absence of any substrate. HPLC-MS was used to confirm that the products of the reactions were lN-methyl-ip or its phosphorylated derivatives. For the HPLC-MS (ESI) analysis, samples containing phosphorylated ip nucleotides were first treated with alkaline phosphatase to remove the phosphate. Two ion transitions were monitored to detect >P 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. Figs. 4A to 4C are mass chromatograms of reaction mixtures prepared using protein Nl, after treatment with alkaline phosphatase. The chromatograms show ion transitions 259.1 [M+H]+ -> 139.1 and 259.1 [M+H]+ -> 169.1 with retention times of 3.4 to 3.629 min, which correspond to lN-methylpseudouridine. Fig. 4A shows results obtained using pseudouridine as the substrate; Fig. 4B shows results obtained using pseudouridine-5'-monophosphate as a 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 lN-methyl-ip. As may be seen, the retention time and 258.0852 Da molar mass of 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 with commercially available standard and observing the area increase of target peaks for ion m / z 139.1 (ion transition 259.1 [M+H]+ -> 139.1) and 169.1 (ion transition 259.1 [M+H]+ -> 169.1). The results of tests performed using enzyme Nl 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 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. 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 lN-methyl-UJ in the sample was calculated by comparing area of IN-methyl-UJ peak in the measured samples to the area of lN-methyl-UJ peak in a standard samples with known concentration. In the most efficient reaction of those performed in this study, the yields of IN-methyl-UJ, lN-methyl-iPMP and lN-methyl-tPTP were approximately 37 %; 36 % and 37 % respectively. The table belowshowsthe measured level of activity of selected enzymes. The list of enzymes is not exhaustive. At least 42 further enzymes which were active on some or all of the substrates were identified in the present study. Protein (SEQ ID NO) Activity on Substrate ip LUMP IPTP N1 (SEQ ID NO: 1) +++ ++ ++ N2 (SEQ ID NO: 2) n.d. ++ +++ El (SEQ ID NO: 9) +++ + ++ E2 (SEQ ID NO: 10) +++ ++ ++ E3 (SEQ ID NO: 11) ++ ++ +++ E4 (SEQ ID NO: 12) +++ +++ +++ Key: N: natural enzyme E: engineered enzyme n.d: not determined +: low relative activity ++: medium relative activity +++: 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-Nl) methyltransferase derived from Methanocaldococcus jannaschii. It is a dimeric protein, belonging to the SPOUT superfamily 5 of SAM-dependent methyltransferases. The dimer includes an a / p knot structural domain that forms part of the SAM binding pocket. N1 has an amino acid sequence of: MREFIFKANKTITSSDINLKDLPGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCIK 10 FVGSELKKVSPDERNIAIFIKKALKKFEELDEEQRKDWNQSTPGIYVRRLGFRNLVLEKLEEGKNIY YLHMNGEDVENVDIENPVFIIGDHIGIGEEDERFLDEIKAKRISLSPLELHANHCITIIHNVLDKKRI CEI (SEQID NO: 1) Residues R28, D30, and R34 were identified as active site residues. 15 Residue H185 was identified as a substrate binding residue that forms a stacking interaction with the pseudouridine base. The following mutations to SEQ ID NO: 1 were identified as potentially beneficial: Mutation Observations K10Q Improves activity when combined with K20D, K75N and optionally (i) R81L or(ii) K74D and K89N. K10Q. alone did not result in a measurable change in activity. It is believed that K10 pulls the substrate away from the catalytic 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 derivatives by forming a salt bridge to the phosphate group. S77R Increases affinity for phosphorylated pseudouridine derivatives by forming a salt bridge to the phosphate group. S77K provides a larger improvement. P23D Improves orientation of positive charge from position 77 but potentially reduces protein solubility. P23K Increases affinity for phosphorylated pseudouridine derivatives by forming a salt bridge to the phosphate group. S77D Useful in combination with P23K. May orient P23K in place. The following mutants of protein N1 were investigated: ID Mutations Observations El K10Q, K20D, K75N Improvement in activity compared to WT E2 K10Q, P23D, S77K, D79N Improvement in activity compared to WT. More active than N2. E9 K10Q, K20N, K75N, R81L Improvement in activity compared to WT E10 K10Q, K20D, K75N, K74D, K89N Improvement in activity compared to WT Ell K10Q Approximately the same the activity as WT E5 D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, 1193V, K199R, K200R, R201E, C203F Most active mutant of SEQ ID 1 out of those tested. E12 D104Y, R115K, N121D, V123I, M138K, N139D, I155L, R167K, R176K, K200R, R201N, C203del, E204del,and l205del Second most active mutant of SEQ ID NO: 1 E7 L32V, D38S, Q59E, C65A, S71N, E100N, Q101K, K103E, D104E, N106T, Q107E, R115S, R116K, Third most active variant of SEQ ID NO: 1 L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, 1154V, I155L, R167K, D170E, K173G, R176K, 1177V, N187D, 1193V, K200R, R201G, C203del, E204del, and l205del El had the following sequence: MREFIFKANQTITSSDINLDDLPGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCI KFVGSELKNVSPDERNIAIFIKKALKKFEELDEEQRKDWNQSTPGIYVRRLGFRNLVLEKLEEGKNI YYLHMNGEDVENVDIENPVFIIGDHIGIGEEDERFLDEIKAKRISLSPLELHANHCITIIHNVLDKKR ICEI (SEQID NO: 9) E2 had the following sequence: MREFIFKANQTITSSDINLKDLDGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCI KFVGSELKKVKPNERNIAIFIKKALKKFEELDEEQRKDWNQSTPGIYVRRLGFRNLVLEKLEEGKNI YYLHMNGEDVENVDIENPVFIIGDHIGIGEEDERFLDEIKAKRISLSPLELHANHCITIIHNVLDKKR ICEI (SEQID NO: 10) E5 had the following sequence: MREFIFKANKTITSSDINLKDLPGSCGRLDLLCRCVSSAFFLSHDIRRDVVFYAVLYGQPNPPVALK FVGSELKKVSPDERNIAIFIKKALKKFEELDEEQRKDWNESTPGIYVRRLGFRNLVLEKLEEGKNIY YLHMNGEDVEEVDIKNPVFILGDHIGIGEEDERFLDEIKAKKISLSPLELHANHCITIVHNVLDRREI FEI (SEQID NO: 15) E12 had the following sequence: MREFIFKANKTITSSDINLKDLPGSCGRLDLLCRCVSDAFFLSHDIRRDVVFYAVLYGQPNPPVCIK FVGSELKKVSPDERNIAIFIKKALKKFEELDEEQRKYWNQSTPGIYVKRLGFRDULEKLEEGKNIYY LHKDGEDVENVDIENPVFILGDHIGIGEEDEKFLDEIKAKKISLSPLELHANHCITIIHNVLDKRNI (SEQID NO: 13) E7 had the following sequence: MREFIFKANKTITSSDINLKDLPGSCGRLDLVCRCVSSAFFLSHDIRRDVVFYAVLYGEPNPPVAIK FVGNELKKVSPDERNIAIFIKKALKKFEELDENKREEWTESTPGIYVSKKGFRDULEKKKEGKKIYYL HKDGEDIENVKINENENPVFVLGDHIGIGEEDEKFLEEIGAKKVSLSPLELHADHCITIVHNVLDKR Gl (SEQID NO: 14) Protein N2 Protein N2 is a tRNA (pseudouridine54-Nl)-methyltransferase derived from Thermococci archaeon. N2 is a dimeric protein, belonging to the SPOUT superfamily of SAM-dependent methyltransferases. The dimer includes an a / p knot structural domain that forms part of the SAM binding pocket. Protein N2 has an amino acid sequence of: MVVFIVKSNTAKTKFLLKDLPGSGKRIDILCRCVNSAFCLSHDIRKDVILYLCFAGKTIKFVGKELKH LTPDERGIAIURKALEGNPTPGVYVSEKSFQDTLLESGKEIIYLDERGEDISHLKLKKDMCFVLGDH LGFDREDQKILEKIGKKISISPKILHADHCIIVVHNFLDRS (SEQID NO: 2) 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 rRNAsmall subunit pseudouridine methyltransferase Nepl from Saccharomyces cerevisiae. N3 is a dimeric protein belonging to the SPOUT superfamily of SAM-dependent methyltransferases. The dimer includes an a / p knot structural domain that forms part of the SAM binding pocket. Protein N3 has an amino acid sequence of: MVEDSRVRDALKGGDQKALPASLVPQAPPVLTSKDKITKRMIVVLAMASLETHKISSNGPGGD KYVLLNCDDHQGLLKKMGRDISEARPDITHQCLLTLLDSPINKAGKLQVYIQTSRGILIEVNPTVRI PRTFKRFSGLMVQLLHKLSIRSVNSEEKLLKVIKNPITDHLPTKCRKVTLSFDAPVIRVQDYIEKLDD DESICVFVGAMARGKDNFADEYVDEKVGLSNYPLSASVACSKFCHGAEDAWNIL (SEQ ID NO: 3) 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 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. Mutation Observations K106Q Reduces non-productive binding of substrate. R129N Forms a hydrogen bond to the ribose group of the substrate. S233W Improves substrate binding by forming a stacking interaction with the pseudouridine base. R132S Makes space for S233W and helps to orient catalytic 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 YVLLNCDDHQGLLKKMGRDISEARPDITHQCLATLLASPINKAGKLQVYIQTSRGILIEVNPTVRIP STFKRFSGLMVQLLHKLSIRSVNSEEKLLKVIKNPITDHLPTKCRKVTLSFDAPVIRVQDYIEKLDDD ESICVFVGAMARGKDNFADEYVDEKVGLSNYPLWASVACSKFCHGAEDAWNIL (SEQ ID NO: 11) Protein N4 5 Protein N4 is an rRNA small subunit pseudouridine methyltransferase Nepl from Candidatus Verstraetearchaeota archaeon. N4 is a dimeric protein belonging to the SPOUT superfamily of SAM-dependent methyltransferases. The dimer includes an a / p knot structural domain that forms part of the SAM binding pocket. 10 Protein N4 has an amino acid sequence of: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDISLHYEAMKKLPNFEKRGRPDII HTTLLTILGSPANLEGLVRTYIHTINDQVVYIDPSVKIPRNYNRFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFQKGSFLKKHVELADK 15 VYAVYGSPLDTWVIASMLLHGYEIEKGII (SEQ ID NO: 4) Residues R63, D65, and R107 were identified as active site residues. The following mutations to SEQ ID NO: 4 were identified as beneficial: Mutation Observations D210H Improves substrate affinity by forming a stacking interaction with the pseudouridine base when combined with a further mutation to provide space for this interaction (e.g., R107S and / or W212H). D210W Improves substrate affinity by forming a stacking interaction with the pseudouridine base. D210Y Improves substrate affinity by forming a stacking interaction with the pseudouridine base. Provides a larger improvement in activity than D210H or D210W. R107S Makes space for stacking from D210 position and helps to orient R63 by hydrogen bonding to D65. W212A Makes space for stacking from D210 position. W212H Makes space for stacking from D210 position. Provided a greater improvement than W212A for mutants containing D210H. W212N Makes space for stacking from D210 position. Provided a greater improvement than W212A for mutants containing D210Y. I45K May improve affinity for phosphorylated substrates by forming a salt bridge. Provided little effect on activity when used alone. A synergistic improvement in activity was observed when this mutation 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-productive binding. K187H May reduce non-productive binding. Q186A Increases activity when combined with D210Y. G76A / N A G76A or G76N mutation was present in all of the variants that included a mutation at position D210. However, the effect of G76A / N was not entirely clear, and this mutation is not believed to be essential. 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, 5 G160E, E161N, K162G, I163E, N164K, S166N, T167I, L168S, A169T, K170L, E171A, L172K, C173E, Q174L, E175C, N176Q, R177E, P178N, T179R, V180P, I181T, 1182V, 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. 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 IHTTLLTILASPANLEGLVRTYIHTINDQVVYIDPSVNIPSNYNQFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFAHGSFLKKHVELADK VYAVYGSPLYTNVIASMLLHGYEIEKGII (SEQ ID NO: 16) Another engineered methyltransferase with an amino acid sequence similar to that of N4 was 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 N4is E4. E4 had the following amino acid sequence: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDKSLHEEAMKKLPNFEKRGRPDI IHTTLLTILASPANLEGLVRTYIHTINDQVVYIDPSVNIPSNYNQFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFQHGSFLKKHVELADK VYAVYGSPLH TAVIASMLLH GYEIEKGII (SEQ ID NO: 12) 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, P150l,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, 1182V, 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, 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 derived from an unidentified microorganism believed to be in the phylum Verstraetearchaeota, having a sequence of: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDISLHYEAMKKLPNFEKRGRPDII HTTLLTILGSPANLEGLVRTYIHTINDQVVYIDPSVKIPRNYNRFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFQKGSFLKKHVELADK VYAVYGSPLDTWVIASMLLHGYEIEKGII (SEQID 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, Y222H, E223,1224G, 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. Example 2. Enzymatic production of Nl-methyl pseudouridine in vivo E. coli cells were transformed by recombinant plasmids harbouring Nl, N2, E3 enzymes or catalytic Nl mutant (negative control) by electroporation. Transformants were grown overnight at 37 °C on LB-agar plates (50 pg / mL ampicillin), suspended in 20 mL liquid LB-medium (50 pg / mL ampicillin) and grown in a shaker at 37°C until ODeoo reached 0.6 - 0.8. After reaching the desired ODeoo, 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 pL of the same buffer that contained 5 mM of pseudouridine (ip). 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 pL of 50 mM potassium phosphate buffer (pH 7.5) and lysed by sonication. The cell remnants were removed by centrifugation. 5 pL of FastAP were added to 50 pL of each reaction mixture, mixtures were incubated at 37 °C overnight. 50 pL 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. Nl-methyl pseudouridine concentration was determined according to the calibration curve prepared from the standard. Results are summarized in the table below. Nl-methyl-tp yield, % Nl-methyl-tp, pM 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 Nl-methyl pseudouridine can be produced in vivo using a microorganism that expresses a methyltransferase as provided herein. Example 3. Enzymatic production of Nl-methyl-2'-deoxypseudouridine and its derivatives Reaction mixtures containing 25 mM Tris-HCI (pH 7.5), 100 mM KCI, 300 mM NaCI, ImM MgCh, 0.03 % NP-40, 25 pM S-adenosyl-L-methionine (SAM), 1 mM 2'-deoxypseudouridine substrate (d4J) and 7 pM N1 methyltransferase in a total volume of 25 pL 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 measured SAH concentrations are shown in Fig. 8. The data shown in Fig. 8 demonstrate that enzymes Nl, E5, and E8 were all found to methylate dUJ. 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 dip, dUJMP, and dYTP. E8 had a sequence of: MSALLNVILAESALELVPKSILDHPAVTKNAERRGKKPGDTLLDKSLHEEAMKKLPNFEKRGRPDI IHTTLLTILASPANLEGLVRTYIHTINDQVVYIDPSVNIPSNYNQFVGLMEQLLKEGRVPPKGDLVL MYVKKQSLEGLLKEIKPTRTFMLSENGEKINISTLAKELCQENRPTVIIGGFAHGSFLKKHVELADK VYAVYGSPLWTNVIASMLLHGYEIEKGII (SEQID NO: 17) Example 4. Enzymatic production of Nl-ethylpseudouridine and its derivatives Reaction mixtures containing 25 mM Tris-HCI (pH 7.5), 100 mM KCI, 300 mM NaCI, ImM MgCl2, 0.03 % NP-40, 20 pM S-adenosyl-L-ethionine (SAE), 1 mM pseudouridine substrate (UJ or UJMP) and 6-12 pM methyltransferase (according to the protein) in a total volume of 25 pL 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). Figs. 9Aand 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 YMP, YTP, dip, dMJMP or dYTP. Example 5. Cap methyltransferases Methyltransferases that have no reported activity with pseudouridine, but whose active sites are potentially capable of methylating pseudouridine substrates were identified as follows. 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. 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 off. 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 % NaCI (Roth) and 100 pg / 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 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-HCI (pH 7.5), 5 mM DTT, 0.03 % NP40, 50 pM SAM, 5 mM GTP and 0.3-12 pM enzyme (according to protein) were incubated at 37 °C. The reactions were stopped after 1 h and activity was assessed by detectingS-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. 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 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 and Cap methyltransferases shows that Cap methyltransferases will be able to methylate and ethylate ip, iPMP, IPTP, d% d^PMP, and diPTP. Proteins SI to S4 as discussed below were investigated. Fig. 10 is a pairwise sequence identity matrix of proteins SI to S4. The Fig. demonstrates that, despite all being mRNA cap guanine-N7 methyltransferases, SI to S4 have less than about 40 % sequence identity. Protein SI Protein SI is a Class 1 SAM dependent methyltransferase, more specifically an mRNA cap guanine-N7 methyltransferase from Nematocida parisii having an amino acid sequence of: MEKSNNNVANHYNKIKSLGVQSREASKIIGVREANNFLKQKLIQKFIRENSVVLDLGCGKGGDLS KLKHHNIKHYYGCDIAKESLAEALKRSLTHKFKSDFLQADFINNKIIIQEKADLVMAQFSFHYAFA NENSVKKAVNNVCNNLKEGGVFILTIPDMQVITRRSARNIVDGSFGNSLYKVCPNKSFYKNELFG RGYEFHLQ.EALTGCEEYLIDLNYLTSHFASKGIKKIFDIDFLSFLNHEMSADKETYSRMVRHPLTKE ELPIIELYRAVAYKKNGS (SEQ ID NO: 5) The activity of wild-type protein SI was below the limit of detection of the assays used. However, analysis demonstrated that activity can be enhanced by introducing mutations. 5 The following useful mutations to SEQ ID NO: 5 have been identified: Mutation Effect E212Q, E212N, E212S, E212C, E212A, E212T, E212M, E212H, E212G, E212V, E212L, or E212I. Removal of negative charge from position 212 should stabilize deprotonated pseudouridine base. E212K The introduction of a positive charge at position 212 should stabilize the deprotonated pseudouridine base more effectively than removing the negative charge. A K residue may downshift the pKa of pseudouridine base. E212R Introducing an R residue at position 212 should be even more effective than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases utilize catalytic arginine residues to downshift the pKa of pseudouridine base. Y271RorY217K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. F201RorF201K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. !267Rorl267K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting 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 S2 5 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 LLDIGCGKGGDLNKYNKARIAQLYAFDVASVSIDQATERYRQMHRPWFKAQFQALDCYNDSIE 10 PYMPRGASVGAVSMQFCAHYAFQSEKQVRIMLENVSRWLAPGGYYFGTVPDANVLVRKLRAS PGLEYGNSIYKIRFVQKDAYPVYGHEYSFLLEDAIDDCPEYLIHWPSFVRLAAEYGLEQVEHTNFH PFYHEQADKFRDLLVRMKVVTEDRPELSMDEWEAAGLYSVFVFRKRGS (SEQID NO: 6) The activity of wild-type protein S2 was below the limit of detection of the assays used. 15 However, analysis demonstrated that activity can be enhanced by introducing mutations. The following useful mutations to SEQ ID NO: 6 have been identified: Mutation Effect E226Q, E226N, E226S, E226C, E226A, E226T, E226M, E226H, E226G, E226V, E226L, orE226l. Removal of negative charge from position 226 should stabilize deprotonated pseudouridine base. E226K The introduction of a positive charge at position 226 should stabilize the deprotonated pseudouridine base more effectively than removing the negative charge. A K residue may downshift the pKa of pseudouridine base. E226R Introducing an R residue at position 226 should be even more effective than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases utilize catalytic arginine residues to downshift the pKa of pseudouridine base. Y290RorY290K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. F215RorF215K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. A286RorA286K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting 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 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 sequence of: 10 MSVVNVDQIIRKHYNERTFVAKRRRRHLSPIIKLRNFNNAIKYMLIDKFTFPGNVVLEMGCGKG GDLRKYGAAGISQFIGIDISNASIVEAQKRFSSMGNLDYQVILITGDCFGESLGVAVEPFPECRFPC DVVSAQFCLHYAFESEEKARRTLLNVTKSLKIGGYFIGTIPDSEFIRYKLNKITKDVDKPSWGNAIY KVTFENSDYQKNNNEFTSPFGQMYTYWLEDAIDNVPEYVIPFETLRSLADEYGLELELQMPFNA FFVQEIPKWINI<FSPI<MQEGLQRSDGKYGVEGDEI<EAASYFYTVFAFI<I<VI<GS (SEQ ID NO: 7) 15 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. The following useful mutations to SEQ. ID NO: 7 have been identified: Mutation Effect E235Q, E235N, E235S, E235C, E235A, E235T, E235M, E235H, E235G, E235V, E235L, orE235l. Removal of negative charge from position 235 should stabilize deprotonated pseudouridine base. E235K The introduction of a positive charge at position 235 should stabilize the deprotonated pseudouridine base more effectively than removing the negative charge. A K residue may downshift the pKa of pseudouridine base. E235R Introducing an R residue at position 235 should be even more effective than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases utilize catalytic arginine residues to downshift the pKa of pseudouridine base. Y304R or Y304K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. Y224RorY224K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. A299RorA299K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting 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 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 5 sequence of: MEGKKEEIREHYNSIRERGRESRQRSKTINIRNANNFIKACLIRLYTKRGDSVLDLGCGKGGDLLK YERAGIGEYYGVDIAEVSINDARVRARNMKRRFKVFFRAQDSYGRHMDLGKEFDVISSQFSFHY AFSTSESLDIAQRNIARHLRPGGYFIMTVPSRDVILERYKQGRMSNDFYKIELEKMEDVPMESVR EYRFTLLDSVNNCIEYFVDFTRMVDGFKRLGLSLVERKGFIDFYEDEGRRNPELSKKMGLGCLTRE 10 ESEVVGIYEVVVFRKLVPESDA (SEQ ID NO: 8) 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. 15 The following useful mutations to SEQ ID NO: 8 have been identified: Mutation Effect E210Q, E210N, E210S, E210C, E210A, E210T, E210M, E210H, E210G, E210V, E210L, orE210l. Removal of negative charge from position 210 should stabilize deprotonated pseudouridine base. E210K The introduction of a positive charge at position 210 should stabilize the deprotonated pseudouridine base more effectively than removing the negative charge. A K residue may downshift the pKa of pseudouridine base. E210R Introducing an R residue at position 210 should be even more effective than introducing a K residue provided that the R residue is orientated correctly. tRNA and rRNA SPOUT methyltransferases utilize catalytic arginine residues to downshift the pKa of pseudouridine base. Y269RorY269K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. F199RorF199K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting the pKa of the pseudouridine base. V265RorV265K Introducing a positive charge should stabilize the deprotonated pseudouridine base by downshifting 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. Example 6. in vivo synthesis of lN-methylpseudoridine from a uridine feedstock The methods of synthesising lN-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 lN-methyl pseudouridine using uridine as a starting material. In the reaction scheme of Fig 11, uracil is first cleaved into uracil and ribose-l-monophosphate using uridine phosphorylase (udp) and inorganic phosphate. A phosphopentomutase (deoB) then isomerizes the ribose-l-monophosphate into ribose-5-monophosphate. The ribose-5-monophosphate is then connected to uracil using pseudouridine monophosphate glycosidase (psuG), to obtain pseudouridine 5'-monophosphate. 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 a suitable methyltransferases and S-adenosyl methionine (SAM) as the methyl group donor. This forms the lN-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) 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; ii) pCDF-udp-deoB and pRSF-psuG-B2P23; iii) pCDF-udp-deoB and pRSF-psuG-B5P8; and iv) pCDF-udp-deoB and pRSF-psuG-BHP9. Transformants were grown overnight at 37 °C on LB-agar plates (50 pg / mL spectinomycin and 30 pg / mL kanamycin). Transformants were suspended in 20 mL liquid LB-medium (50 pg / mL spectinomycin and 30 pg / 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, washed with 1 mL of 125 mM potassium phosphate buffer (pH 7.0), and resuspended into 100 pL of the same buffer that contained 5 mM uridine and 0.5 mM MnCL 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 pL of 125 mM potassium phosphate buffer (pH 7.0), and lysed by sonication. Cellular debris were removed by centrifugation. 5 pL of FastAP were added to the tubes, that contained 50 pL of buffer in which reactions were done, or 50 pL of supernatant after cell sonication. The resulting mixtures were incubated at 37 °C and 300 rpm overnight. 50 pL of acetonitrile were added to each tube, mixtures were centrifuged for 10 min at 4 °C at 16 000 rpm to precipitate the remaining proteins. The reaction mixtures were analyzed by HPLC-MS. The lN-methyl pseudouridine (mlMJ) 5 concentration was determined according to the calibration curve prepared from the standard. Nl-Methyl pseudouridine was detected in the reaction buffer where B2P3 methyltransferase was present, however no lN-methyl pseudouridine was detected in analyzed cell content. The final yield of lN-methyl pseudouridine was 0.162 %, corresponding to a lN-methylpseudouriding concentration of 8.1 pM in the reaction mixture. 10 It will be appreciated that the above embodiments have been described by way of example only. Other variants or use cases of the disclosed techniques may become apparent to the person 15 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

1. A method of synthesising a IN-alkyl derivative of pseudouridine, which methodcomprises:contacting a substrate with a methyltransferase in the presence of a cofactor to produce the IN-alkyl derivative of pseudouridine;wherein:the substrate has a structure of:the lN-methyl derivative of pseudouridine has a structure of:R1 being a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group;R2 being H or a hydroxyl group; andR3 being a methyl group or an ethyl group;the cofactor is a source of a methyl group or an ethyl group; andthe 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 the cofactor is S-adenosyl-L-methionine orS-adenosyl-L-ethionine.

3. The method according to claim 2, wherein R3 is a methyl group and the cofactor is S-adenosyl-L-methionine.

4. The method according to any preceding claim, wherein R1 is a hydroxyl group, a monophosphate group, or a triphosphate group.

5. The method according to claim 4, wherein R1 is a hydroxyl group or a monophosphate group, and the method further comprises, after the contacting, phosphorylating the lN-alkyl derivative of pseudouridine.

6. The method according to any preceding claim, wherein R2 is a hydroxyl group.

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

8. The method according to claim 7, further comprising contacting the pseudouridine 5'-monophosphate with a phosphatase to form pseudouridine.

9. The method according to claim 7 or claim 8, further comprising forming the D-ribose-5-monophosphate by contacting a-D-ribose-l-monophosphate with a phosphopentomutase.

10. The method according to claim 9, further comprising forming the D-ribose-5-monophosphate by contacting uridine with a uridine phosphorylase and an inorganic phosphate.

11. 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.

12. The method according to claim 11, wherein the contacting is performed in the presence of the microorganism; optionally wherein the method is performed in vivo.

13. 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.

14. The method according to claim 13, wherein the buffer has a pH in the range 7.4 to 7.6.

15. 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 04 of the substrate and / or the active site has a net positive charge of one or two.

16. 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.

17. The method according to claim 16, wherein the methyltransferase is a cap methyltransferase, optionally an mRNA cap guanine-N7 methyltransferase.

18. The method according to claim 17, 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).

19. The method according to claim 17, 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) F215Ror F215K;ii) E226a E226K, E226R, E226N, E226S, E226C, E226A, E226T, E226M, E226H, E226G, E226V, E226L, or E226I;iii) A286R or A286K;iv) Y290R orY290K;optionally wherein the amino acid sequence includes no more than two mutations selected from i) to iv).

20. The method according to claim 17, 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 orY304K;optionally wherein the amino acid sequence includes no more than two mutations selected from i) to iv).

21. The method according to claim 17, 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; andiv) Y269R or Y269K;optionally wherein the amino acid sequence includes no more than two mutations selected from i) to iv).

22. The method according to any of claims 1 to 15, wherein the methyltransferase is a dimeric SPOUT methyltransferase comprising a SPOUT domain configured to transfer the methyl group from the cofactor to the substrate.

23. The method according to claim 22, wherein the methyltransferase is a minimalist dimeric SPOUT methyltransferase, free of any domains other than the SPOUT domain.

24. The method according to claim 23, wherein the methyltransferase consists of the SPOUT domain.

25. 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.

26. The method according to claim 25, 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.

27. The method according to any preceding claim, wherein R1 is 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; andoptionally wherein the amino acid residue which forms the salt bridge is a lysine or arginine residue.

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

29. The method according to any of claims 22 to 28, wherein the methyltransferase is a tRNA (pseudouridine54-Nl)-methyltransferase.

30. The method according to claim 29, wherein the methyltransferase is an engineered tRNA (pseudouridine54-Nl)-methyltransferase which includes at least one of the following modifications compared to wild-type tRNA (pseudouridine54-Nl)-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 / orii) 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.

31. The method according to claim 30, wherein the methyltransferase has an amino acid sequence having at least 70 % sequence identity to SEQ ID NO: 1.

32. The method according to claim 31, wherein the amino acid sequence includes mutations K10Q, K75N, and one of K20D and K20N.

33. The method according to claim 31 or claim 32, wherein the wherein the amino acid sequence includes one or more mutations selected from K74D, R81L, and K89N.

34. The method according to any of claims 31 to 33, wherein the amino acid sequence includes one or more mutations selected from:D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, 1193V, K199R, K2OOR, R201E, and C2O3F.

35. The method according to any of claims 31 to 34, 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 l205del.

36. The method according to any of claims 31 to 35, wherein the amino acid sequence includes one or more mutations selected from:L32V, D38S, Q59E, C65A, S71N, E100N, Q101K, K1O3E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N150_P151insE, P151_V152insN, 1154V, I155L, R167K, D170E, K173G, R176K, 1177V, N187D, 1193V, K200R, R201G, C203del, E204del, and l205del.

37. The method according to any of claims 31 to 36, wherein the amino acid sequence includes a P23K mutation.

38. The method according to claim 37, wherein the amino acid sequence further includes an S77D mutation.

39. The method according to any of claims 31 to 38, wherein the amino acid sequence includes an F41N mutation.

40. The method according to any of claims 31 to 39, wherein the amino acid sequence includes one or more mutations selected from K10Q, P23D, S77K, and D79N.

41. The method of claim 31, 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, 1193V, K199R, K2OOR, R2O1E, and C2O3F;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, K2OOR, R201N, C203del, E204del,and l205del; andvii) SEQ ID NO: 1 with mutations L32V, D38S, Q59E, C65A, S71N, EICON, Q101K, K1O3E, D104E, N106T, Q107E, R115S, R116K, L117K, N121D, V123I, L127K, E128K, N132K, M138K, N139D, V143I, D147K, E149_N150insN, N15O_P151insE, P151_V152insN, 1154V, I155L, R167K, D170E, K173G, R176K, 1177V, N187D, 1193V, K2OOR, R201G, C203del, E204del, and l205del.

42. The method according to claim 29 or claim 30, wherein the methyltransferase has an amino acid sequence having at least 70 % sequence identity to SEQ ID NO: 2.

43. The method according to any of claims 22 to 28, wherein the methyltransferase is an rRNA small subunit pseudouridine methyltransferase Nepl.

44. The method according to claim 43, 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; andii) a neutrally-charged or positively charged active site for catalysing transfer of the methyl group or ethyl group from the cofactor to the substrate.

45. The method according to claim 43 or claim 44, 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.

46. The method according to claim 45, wherein the at least one mutation comprises V2_A27del.

47. The method according to claim 45 or claim 46, wherein the one or more mutations comprise at least one of K106Q, R129N, and S233W.

48. The method according to claim 47, 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.

49. The method according to claim 43 or claim 44, 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.

50. The method according to claim 49, 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, 1182V, 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.

51. The method according to claim 50, wherein the mutations further comprise R107S.

52. The method according to claim 50 or claim 51, wherein the mutations further comprise W212A, W212H, or W212N.

53. The method according to claim 52, wherein the mutations comprise:a) D210H and W212H;orb) D210Yand W212N.

54. The method according to any of claims 50 to 53, wherein the mutations further comprise I45K.

55. The method according to claim 54, wherein the mutations further comprise Y49E.

56. The method according to any of claims 50 to 55, wherein the mutations furthercomprise G76A or G76N.

57. The method according to any of claims 50 to 56, wherein the mutations comprise D210Y and Q186A.

58. The method according to any of claims 49 to 57, wherein the mutations comprise one or more of: K104N, R1UQ, and KWH.

59. The method according to claim 53, wherein the mutations are I45K, Y49E, G76A, K104N, R107S, R111Q, Q186A, K187H, D210Y, and W212N.

60. 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, l205del, Q101K, 1154V, E128K, N139D, V123I, L117K, E204del, N121D, R201E, D104Y, C203F, L32V, 1177V, 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, 1193V, Q59E, E100N, K103E, K75N, L127K, D170E, C65A, C203del, K74D, and R167K.

61. The methyltransferase according to claim 60, wherein the mutations are:i) K10Q, K20D, and K75N; orii) K10Q, K20N, K75N, and R81L; oriii) K10Q, K20D, K75N, K74D, and K89N; oriv) D38S, C65A, I66L, Q107E, N145E, E149K, I155L, R176K, 1193V, K199R, K200R, R201E, and C2O3F; orv) K10Q, P23D, S77K, and D79N; orvi) D104Y, R115K, N121D, V123I, M138K, N139D, 1155L, R167K, R176K, K200R, R201N, C203del, E204del, and 1205del; orvii) 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, 1154V, I155L, R167K, D170E, K173G, R176K, 1177V, N187D, 1193V, K200R, R201G, C203del, E204del, and l205del.

62. 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.

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

64. A methyltransferase having an amino acid sequence of SEQ. ID NO: 4 with a mutation selected from D210H, D210Y, D210P, and D210W; andoptionally 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, 1182V, 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.

65. The methyltransferase according to claim 64, wherein the mutations comprise:a) D210H and W212H; orb) D210Yand W212N.

66. The methyltransferase according to claim 64 or claim 65, wherein the mutations include I45K and Y49E.

67. The methyltransferase according to any of claims 64 to 66, wherein the mutations include G76A or G76N.

68. The methyltransferase according to claim 64, wherein the mutations are D210Y and Q186A.

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

70. A methyltransferase having an amino acid sequence of SEQID 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.

71. The methyltransferase according to claim 70, having an amino acid sequence of SEQ ID NO: 12.

72. A methyltransferase having an amino acid sequence of SEQ ID NO: 5 with at least onemutation selected from:i) F2O1R 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.

73. A methyltransferase having an amino acid sequence of SEQ. ID NO: 6 with at least one mutation selected from:i) F215Ror F215K;ii) E226Q, E226K, E226R, E226N, E226S, E226C, E226A, E226T, E226M, E226H,E226G, E226V, E226L, or E226I;iii) A286R or A286K;iv) Y290R orY290K.

74. A methyltransferase having an amino acid sequence of SEQ ID NO: 7 with at least onemutation 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 orY304K.

75. 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, E21OR, E210N, E210S, E21OC, E210A, E210T, E210M, E21OH,E210G, E210V, E21OL or E210I;iii) V265R or V265K;iv) Y269R or Y269K.

76. The methyltransferase of any of claims 72 to 75, wherein the amino acid sequence includes no more than two mutations.

77. A polynucleotide having a sequence encoding a methyltransferase as defined in any of claims 60 to 76.

78. An expression vector carrying the polynucleotide of claim 77.

79. A microorganism transduced with the expression vector of claim 78.

80. 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 59, 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.

81. Use according to claim 80, wherein the aqueous buffer is a Tris buffer.

82. Use according to claim80 or claim 81, wherein the aqueous buffer includes NaCI at a concentration of 50 to 350 mM, and KCI at a concentration of 70 to 130 mM.

83. Use according to any of claims 80 to 82, wherein the Mg2+ is supplied by MgCh.

84. Use according to any of claims 80 to 83, wherein:the cofactor is present in the aqueous buffer at a concentration in the range 20 to 30 pM; and / orthe substrate is present in the aqueous buffer at a concentration in the range 80 to 120 pM; and / orthe methyltransferase is present in the aqueous buffer at a concentration in the range 0.5 to 20 pM.

85. Use according to claim 84, wherein the buffer has a pH of 7.5 ± 0.1 and includes:Tris-HCI at a concentration of about 25 mM;NaCI at a concentration of about 300 mM;MgClz at a concentration of about 1 mM;KCI at a concentration of about 100 mM;the cofactor at a concentration of about 25 pM; andthe substrate at a concentration of about 100 pM.

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