Antifungal polyene macrolides

Abstract

The disclosure provides a polyene. The polyene comprises two sugar moieties and / or is modified in an exocyclic carboxylic acid position, or is a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof. In some embodiments, the polyene is a compound of formula I. The disclosure extends to pharmaceutical and agrochemical compositions, preservatives and foodstuffs comprising the polyene. The disclosure also extends to the polyene or pharmaceutical composition for use as a medicament, for use in treating, preventing or ameliorating an infection or a prion disease and methods of treating a disease in a plant or a mushroom using the polyene. The disclosure further provides a method of producing a polyene comprising a sugar moiety and / or a modification at an exocyclic carboxylic acid thereon. Finally, the disclosure also extends to a recombinant organism or cell expressing a glycosyltransferase enzyme or an amidotransferase enzyme.

Description

[0001] 126210PCT1 - 1 - Polyenes The present invention relates to novel polyene compounds. The invention extends to pharmaceutical compositions, agrochemical compositions and preservatives 5 comprising the novel polyene compounds, and medical, agrochemical and preservative uses thereof. The invention also extends to enzymes which may be used to produce the novel polyenes and methods of producing the polyenes. Polyenes are polyketide macrolide natural products, derived from Streptomyces and 10 related Actinobacteria. Members of the polyene family are known to possess broad spectrum antifungal activity. The clinically important polyenes include amphotericin B (AmB), nystatin (Nys) and pimaricin (Pim), which are all WHO’s essential medicines.1Fungal infections are a serious threat to human health, especially immune- compromised individuals.2-6According to the CDC report, multidrug resistant 15 pathogens (Candida auris, Candida albicans and Aspergillus fumigatus) are a rising global threat. Despite the acute need for alternative treatments, very few new drug candidates have emerged in recent years.7,8Given polyenes have potent broad- spectrum activity and the incidence of resistance to them is relatively low, they remain the most effective and widely used antifungal agents.1For example, AmB is used in 20 chronic aspergillosis where patients fail azole therapy and is also used as an effective treatment for cryptococcosis and black fungus (mucormycosis). In addition to fungal infections, AmB is used against leishmaniasis, a protozoal infection, infecting millions with >20,000 deaths annually.3,9Nys is used to treat Candida infections and as a prophylactic treatment to prevent candidiasis in high-risk patients. Pim is used to treat 25 fungal eye infection, keratitis, a leading cause of blindness. Despite the medical importance, use of polyenes is limited in mammalian cells by dose dependent toxicity arising from non-specific cholesterol binding as well as poor serum solubility.10To address the need for safer alternatives, new improved synthetic polyene 30 derivatives such as AmB-disaccharide methyl ester (MFAME), AmB-urea derivatives and AmB lacking mycosamine C2’ OH have been produced.11-13However these derivatives require multistep syntheses, deleterious reagents and extensive use of protecting groups, which is expensive, unsustainable and difficult to scale-up.14-16Alternative biosynthetic routes to polyene derivatives are more desirable. Polyene 35 macrolides are assembled by polyketide synthase (PKS) enzymes, followed by a series of tailoring steps. For AmB, tailoring involves oxidation of a C16-methyl group to C16- 126210PCT1 - 2 - CO2H by a P450 (AmphN), with subsequent C19-glycosylation (AmphDI) and then C8- hydroxylation by another P450 (AmphL). Analogous tailoring steps occur in other polyene pathways.17,18This knowledge enabled pathway engineering to produce AmB variants, including deactivating domains within the PKS to change oxidation levels of 5 the macrolide.19Deleting enzymes, such as AmphN generates AmB with C16-methyl rather than C16-CO2H.20The addition of tailoring enzymes has also been explored, including heterologous expression of glycosyltransferase (NypY) which led to AmB with a second sugar (mannose) attached to C19-mycosamine, which has improved solubility and reduced toxicity.21Similar approaches have also been used to engineer pathways to 10 Nys,22,23Pim24-26and other important polyenes.26In addition to healthcare, polyenes have commercial applications as food preservatives (pim E235), agrochemicals (pesticides) and in veterinary medicine (Mycophyl®, Panalog®).27, 28The invention arose due to the inventors’ work seeking to develop new enzymatic and 15 biosynthetic methods for the more efficient and sustainable production of improved polyene antimicrobial agents. In accordance with a first aspect of the invention, there is provided a polyene, wherein the polyene comprises two sugar moieties and / or is modified in an exocyclic carboxylic 20 acid position, or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof, wherein the polyene is not: , , 126210PCT1 - 3 - Advantageously, modifying a polyene in exocyclic carboxylic acid position can improve 5 significantly antifungal activity and reduce toxicity of polyenes, whilst addition of a sugar improves solubility. A polyene which is modified in an exocyclic carboxylic acid position may be understood to comprise a CONHR5, CONHNHR5or CONHOR5group, wherein R5is H, a C1-6 alkyl,10a C6-12 aryl or a 5 to 10 membered heteroaryl. Preferably, the polyene is a compound of formula I: 15 wherein L1is an optionally substituted C2-15 alkylene or an optionally substituted C2-15 alkenylene; L2is absent or is an optionally substituted C1-8alkylene or an optionally substituted C2-8alkenylene; 126210PCT1 - 4 - L3is absent or is , where an asterisk indicates a point of bonding to the carbon atom to which the R1group is bonded; R1is H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl or an 5 optionally substituted C3-12 cycloalkenyl; R2is H, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl or an optionally substituted C3-12 cycloalkenyl; R3is an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an10optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12cycloalkenyl, COOR5, CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor CONR5OOR5a; R4is an optionally substituted sugar moiety; R5, R5aand R5bare each independently H, an optionally substituted C1-16 alkyl, an 15 optionally substituted C2-16alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, COR8, CONHR8, a C6-12 aryl or a 5 to 10 membered heteroaryl; R6is H, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an 20 optionally substituted C3-12 cycloalkenyl, an optionally substituted C6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl; R7is OH or an optionally substituted sugar moiety; and R8is H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl or an 25 optionally substituted C3-12 cycloalkenyl; wherein if R3is an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12 cycloalkenyl or COOH then L3is present and R7is an optionally substituted sugar moiety; 30 or a pharmaceutically acceptable salt or solvate thereof; wherein the compound of formula I is not: 126210PCT1 - 5 - 5 Pharmaceutically acceptable salts include any salt of a compound of formula (I) provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. The pharmaceutically acceptable salt may be derived from a variety of organic and inorganic counter-ions well known in the 10 art. The pharmaceutically acceptable salt may comprise an acid addition salt formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic,15cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, 126210PCT1 - 6 - picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2- ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4- methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic, 3-phenylpropionic, 5 trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids. Alternatively, the pharmaceutically acceptable salt may comprise a base addition salt formed when an acidic proton present in the parent compound is either replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, an aluminium 10 ion, alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminium, lithium, zinc, and barium hydroxide, or coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline,15 N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)- aminomethane, tetramethylammonium hydroxide, and the like. A pharmaceutically acceptable solvate refers to a compound of formula (I) provided 20 herein, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate. The term “alkyl” as used herein, unless otherwise specified, refers to a saturated, 25 straight or branched hydrocarbon. The alkyl may be a primary, secondary, or tertiary hydrocarbon. An alkyl, unless otherwise specified, may be a C1-16 alkyl, more preferably a C1-12 alkyl or a C1-6 alkyl. The term “alkenyl” as used herein, unless otherwise specified, refers to an olefinically 30 unsaturated, straight or branched hydrocarbon. Accordingly, an alkenyl group may be understood to be a hydrocarbon which contains one or more double bonds. An alkenyl group may be partially saturated, i.e. it may contain two or more adjacent single bonds. The alkenyl may be a primary, secondary, or tertiary hydrocarbon. An alkenyl, unless otherwise specified, may be a C2-16 alkenyl, more preferably a C2-12 alkenyl or a C2-6 35 alkenyl. 126210PCT1 - 7 - The term “alkynyl” as used herein, unless otherwise specified, refers to an acetylenically unsaturated, straight or branched hydrocarbon. Accordingly, an alkynyl group may be understood to be a hydrocarbon which contains one or more triple bonds. An alkynyl group may be partially saturated, i.e. it may contain two or more adjacent single bonds. 5 For the avoidance of doubt, an alkynyl group may also contain one or more double bonds. The alkynyl may be a primary, secondary, or tertiary hydrocarbon. An alkynyl, unless otherwise specified, may be a C2-16 alkynyl, more preferably a C2-12 alkynyl or a C2-6 alkynyl. 10 The term “alkylene”, as used herein, unless otherwise specified, refers to a bivalent saturated hydrocarbon. The alkylene group is preferably a straight hydrocarbon. The term “alkenylene”, as used herein, unless otherwise specified, refers to a bivalent olefinically unsaturated hydrocarbon. The alkenylene group is preferably a straight 15 hydrocarbon. Accordingly, an alkenylene group may be understood to be a bivalent hydrocarbon which contains one or more double bonds. An alkenylene group may be partially saturated, i.e. it may contain two or more adjacent single bonds. “Cycloalkyl” refers to a non-aromatic, saturated, monocyclic, bicyclic or polycyclic 20 hydrocarbon ring system. A cycloalkyl, unless otherwise specified, may be a C3-12cycloalkyl, more preferably a C5-10cycloalkyl or a C5-7cycloalkyl. Representative examples of a cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. 25 “Cycloalkenyl” refers to a non-aromatic, unsaturated, monocyclic, bicyclic or polycyclic hydrocarbon ring system. Accordingly, a cycloalkenyl group may be understood to contain one or more double bonds. A cycloalkenyl, unless otherwise specified, may be a C3-12cycloalkenyl, more preferably a C5-10cycloalkenyl or a C5-7cycloalkenyl. 30 The terms “heterocycle” and “heterocyclyl” refer to a monocyclic, bicyclic or bridged molecules in which at least one ring atom is a heteroatom. The or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen. A heterocycle, unless otherwise stated, may be a 3 to 8 membered heterocycle. A heterocycle may be saturated or partially saturated. 35 126210PCT1 - 8 - The term “aryl” refers to an aromatic 6 to 12 membered hydrocarbon group. An aryl may be monocyclic, bicyclic or multicyclic. The term “aryl” may be understood to encompass bicyclic ring systems where one of the rings is aromatic and one of the rings is unsaturated or partially saturated and multicyclic ring systems where at least one of 5 the rings is aromatic and at least one of the rings is unsaturated or partially saturated. Examples of a C6-C12 aryl group include, but are not limited to, phenyl, α-naphthyl, β- naphthyl, biphenyl, tetrahydronaphthyl and indanyl. The term “heteroaryl” refers to an aromatic 5 to 10 membered ring system in which at 10 least one ring atom is a heteroatom. A heteroaryl may be monocyclic, bicyclic or multicyclic. In bicyclic and multicyclic structures, a group may be understood to be a heteroaryl if at least one of the rings comprises a heteroatom. The term “heteroaryl” may be understood to encompass bicyclic ring systems where one of the rings is aromatic and one of the rings is unsaturated or partially saturated and multicyclic ring 15 systems where at least one of the rings is aromatic and at least one of the rings is unsaturated or partially saturated. The or each heteroatom may be independently selected from the group consisting of oxygen, sulphur and nitrogen. Examples of 5 to 10 membered heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, 20 pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1- methyl-1,2,4-triazole, 1H-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline and isoquinoline. Bicyclic 5 to 10 membered heteroaryl groups include those where a phenyl, pyridine, pyrimidine, 25 pyrazine or pyridazine ring is fused to a 5 or 6-membered monocyclic heteroaryl ring. Where an alkyl, alkenyl or alkynyl is defined as being optionally substituted it may be substituted or unsubstituted. If substituted the alkyl, alkenyl or alkynyl may be substituted with one or more substituents selected from the group consisting of halo, an 30 optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12 aryl, an optionally substituted 5 to 10 membered heteroaryl, oxo, -O-, hydroxy, a sugar moiety, OR9, COOR9, NR9R10, CN, N3, NO2, SR9, SOR9and SO2R9, wherein R9and R10are independently H, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 35 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12cycloalkenyl, an optionally substituted 3 to 8 membered 126210PCT1 - 9 - heterocycle, an optionally substituted C6-12aryl or an optionally substituted 5 to 10 membered heteroaryl. Where an alkylene, alkenylene, cycloalkyl, cycloalkenyl, heterocycle, aryl, heteroaryl 5 group or sugar moiety is defined as being optionally substituted it may be substituted or unsubstituted. If substituted the alkylene, alkenylene, cycloalkyl, cycloalkenyl, aryl, heteroaryl group or sugar moiety may be substituted with one or more substituents selected from the group consisting of halo, an optionally substituted C1-16 alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16alkynyl, an optionally 10 substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12 aryl, an optionally substituted 5 to 10 membered heteroaryl, oxo, -O-, hydroxy, a sugar moiety, OR9, COOR9, NR9R10, CN, N3, NO2, SR9, SOR9and SO2R9, wherein R9and R10are independently H, a C1-6 haloalkyl, an optionally substituted C1-16 alkyl, an optionally 15 substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12aryl or an optionally substituted 5 to 10 membered heteroaryl. 20 When a group is substituted with an -O- group, the oxygen may be understood to bond to two adjacent carbons to form an epoxy ring. “Halogen” or “halo” may be understood be to F, Cl, Br or I. 25 It will be appreciated that an optionally substituted C1-16alkyl may be an optionally substituted C1-16 haloalkyl, i.e. a C1-16 alkyl substituted with at least one halogen, and optionally further substituted with one or more further substituents, as defined above. The optionally substituted C1-16haloalkyl may be a halomethyl, for instance CF3. 30 A sugar moiety may be a monosaccharide, a disaccharide, a polysaccharide or a derivative thereof. A sugar moiety may be understood to have general formula: 126210PCT1 - 10 - , wherein X1is a bond or CR18R19; R15to R23are each independently H, OR24, COR24, COOR24, NR24R25, N+R24R25R26, NR24COR25, NR24COOR25, NR24CONR25R26, optionally substituted C1-6alkyl, optionally 5 substituted C2-6alkenyl, optionally substituted C2-6alkynyl, halo and R24to R26are independently H, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12cycloalkenyl, an optionally 10 substituted 3 to 8 membered heterocycle, an optionally substituted C6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl. It may be appreciated that when one , then the sugar moiety may be a disaccharide. Alternatively, where there are multiple 15structures in in the compound then the moiety may be a polysaccharide. In some embodiments one or more of R15to R23is an optionally substituted C1-6 alkyl, an optionally substituted C2-6 alkenyl or an optionally substituted C2-6 alkynyl, and more preferably is an optionally substituted C1-3 alkyl, an optionally substituted C2-3 alkenyl 20 or an optionally substituted C2-3alkynyl. In some embodiments, one or more of R15to R23is an optionally substituted methyl, an optionally substituted ethyl or an optionally substituted propyl. 126210PCT1 - 11 - In embodiments where one or more of R15to R23is an optionally substituted C1-6alkyl, an optionally substituted C2-6 alkenyl or an optionally substituted C2-6 alkynyl, the alkyl, alkenyl or alkynyl may be unsubstituted or substituted with one or more constituents as defined above. In some embodiments, the alkyl, alkenyl or alkynyl is unsubstituted or 5 substituted with one or more constituents halo, oxo, OR9, COOR9, NR9R10, N+R9R10R10a, NR9COR10, NR9COOR10, NR9CONR10R10, CN, N3, NO2, SR9, SOR9and SO2R9, wherein R9, R10and R10aare independently H, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12cycloalkenyl, an optionally 10 substituted 3 to 8 membered heterocycle, an optionally substituted C6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl. More preferably, the alkyl, alkenyl or alkynyl is unsubstituted or substituted with one or more constituents halo, oxo, OH, COOH or NH2. 15 In some embodiments, R15to R23are independently H, OH, OCH3, CH3, CH2OH, CH(OH)CH3, CH(OH)CH(OH)CH2OH, COH, COOH, NH2, N(CH3)2 or NHCOCH3. Examples of sugar moieties include glucose, galactose, mannose, fucose, fructose, rhamnose, digitoxose, mycarose, 4-O-methyldigitoxose, ribose, 2-N-acetyl-20 galactosamine, glucuronic acid, N-acetyl-neuraminic acid, ristosamine, 3-N-methyl-4- O-methyl-ristosamine, fucofuranose, rhamnose, nogalose, streptose, furanose, pyranose, olivose, aryl-C-olivose, noviose, 4-O-methyl-noviose, allose, chalcose, chalcomycin, actinospectose, amicetose, mycinose, chalose, mycarose, cladinose, oleandrose, oliose, olivomose, digitalose, olivomycose, evalose, xylose, pentose, 25 quinovose, arcanose, rhodinose, cinerulose, aculose, lyxose, gulose, colitose, mycaminose, cladinose, desosamine, megosamine, rhodosamine, duanosamine, nogalamine, 2-deoxy-nogalamine, forosamine, daunosamine, acosamine, eremosamine, angolosamine, vicenisamine, dimethyl-eremosamine, vancosamine, mycosamine, or a derivative thereof. A derivative of a monosaccharide, a disaccharide 30 or a polysaccaride may be a monosaccharide, a disaccharide or a polysaccaride where an OH group is replaced with a H, OCH3, CH3, NR9R10or NCOR9, wherein R9and R10are independently H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted 3 to 8 membered 35 heterocycle, an optionally substituted C6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl. For instance, an OH group may be replaced with a H, OCH3, 126210PCT1 - 12 - CH3, NH2or NCOCH3. N-Acetylglucosamine may be understood to be an amide derivative of glucose. Perosamine may be understood to be a derivative of mannose. A sugar moiety can be alpha or beta anomer. A sugar moiety may be an L or D form. 5 The compound of formula (I) may be a compound of formula (Ia): The compound of formula (Ia) may be a compound of formula (Ib): 10 The compound of formula (Ib) may be a compound of formula (Ic): 15 L1may be an optionally substituted C3-12alkylene or an optionally substituted C3-12alkenylene. More preferably, L1is an optionally substituted C4-10 alkylene or an optionally substituted C4-10 alkenylene. Most preferably, L1is an optionally substituted 20 C5-9alkylene or an optionally substituted C5-9alkenylene. 126210PCT1 - 13 - In embodiments where L1is an alkenylene, the alkenylene may contain one double bond. In some embodiments, L1is optionally substituted pentylene, optionally substituted 5 hexylene, optionally substituted heptylene, optionally substituted octylene, optionally substituted nonylene, optionally substituted decylene or optionally substituted undecylene. In some embodiments, L1is optionally substituted pentenylene, optionally substituted hexenylene, optionally substituted heptenylene, optionally substituted octenylene, optionally substituted nonenylene, optionally substituted decenylene or 10 optionally substituted undecenylene. Preferably, L1is a substituted alkylene or a substituted alkenylene. The alkylene or alkenylene group may be substituted with one or more substituents selected from the group consisting of halo, an optionally substituted C1-16 alkyl, an optionally substituted 15 C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12aryl, an optionally substituted 5 to 10 membered heteroaryl, oxo, -O-, hydroxy, a sugar moiety, OR9, COOR9, NR9R10, CONR9R10, CONR9NR10R11, CONR9OR10, CONR9NR10OR11, CONR9OOR10or 20 NR9CONR10R11, CN, N3, NO2, SR9, SOR9and SO2R9, wherein R9, R10and R11are independently H, a C1-6haloalkyl, an optionally substituted C1-16alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12aryl or an 25 optionally substituted 5 to 10 membered heteroaryl. In some embodiments, the alkylene or alkenylene group may be substituted with one or more of OH, -O-, oxo and / or a C1-6 alkyl group. When the alkylene or alkenylene group is substituted with an -O- group, the oxygen may be understood to bond to two adjacent carbons in the alkylene or alkenylene to form an epoxy ring. The C1-6alkyl may be methyl, ethyl or 30 propyl. In some embodiments, L1is a substituted alkylene or a substituted alkenylene, wherein the alkylene or alkenylene is substituted with at least one OH group. The alkylene or alkenylene may be substituted with one, two, three or four OH groups. 35 126210PCT1 - 14 - Alternatively, or additionally, L1may be a substituted alkylene or a substituted alkenylene, wherein the alkylene or alkenylene is substituted with at least one oxo group. The alkylene or alkenylene may be substituted with one or two oxo groups. 5 Alternatively, or additionally, L1may be a substituted alkylene or a substituted alkenylene, wherein the alkylene or alkenylene is substituted with at least one -O- group. The alkylene or alkenylene may be substituted with one -O- group. Alternatively, or additionally, L1may be a substituted alkylene or a substituted 10 alkenylene, wherein the alkylene or alkenylene is substituted with at least one C1-6 alkyl group. The alkylene or alkenylene may be substituted with one C1-6 alkyl group. The C1- 6 alkyl group may be methyl, ethyl or propyl. 15 bonding 20 to the carbon atom to which an OH group is bonded. 126210PCT1 - 15 - 5 asterisk indicates a point of bonding to the carbon atom to which an OH group is bonded. 10 L2may be absent. Alternatively, L2is an optionally substituted C1-8alkylene or an optionally substituted C2-8alkenylene. The alkylene or alkenylene group may be unsubstituted or substituted with one or more substituents selected from the group consisting of halo, an optionally 15 substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12 aryl, an optionally substituted 5 to 10 membered heteroaryl, oxo, -O-, hydroxy, a sugar moiety, OR9, COOR9, NR9R10, CONR9R10, CONR9NR10R11, CONR9OR10, 20 CONR9NR10OR11, CONR9OOR10or NR9CONR10R11, CN, N3, NO2, SR9, SOR9and SO2R9, wherein R9, R10and R11are independently H, a C1-6haloalkyl, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an 126210PCT1 - 16 - optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12aryl or an optionally substituted 5 to 10 membered heteroaryl. Preferably, L2is an C1-8 alkylene or a C2-8 alkenylene. Preferably, L2is a C2-6 alkylene or 5 a C2-6alkenylene. More preferably, L2is a C2-6alkenylene. The alkenylene may contain one, two or three double bonds. Most preferably, L2is a C2 or C6 alkenylene. The C6 alkenylene may contain two or three double bonds. 10 , , where an asterisk indicates a point of bonding to L3, or where L3is absent to the carbon atom to which the R1group is bonded. 15 L3may be absent. Preferably, in embodiments where L2is absent then L3is also absent. In some embodiments where L2a C1-8 alkylene or a C2-8 alkenylene then L3is absent. Alternatively, L3may be . In some embodiments where L2a C1-8alkylene or a C2-8 alkenylene then . 20 L3may . In some embodiments, the compound of formula I may be a compound of formula II, III, IV, V, VI, VII, VIII, IX or X: 5 10

[0002]  126210PCT1 - 19 - (X) wherein R11is H or OH. 5 In some embodiments, the compound of formula II may be a compound of formula IIa: In some embodiments, the compound of formula III may be a compound of formula 10 IIIi or IIIii: 15 In some embodiments, the compound of formula III may be a compound of formula IIIa: 126210PCT1 - 20 - In some embodiments, the compound of formula IIIa may be a compound of formula 5 IIIai or IIIaii: 10 R11may H. In a preferred embodiment, R11is OH. In some embodiments, the compound of formula IV may be a compound of formula 15 Iva: (IVa) 126210PCT1 - 21 - In some embodiments, the compound of formula V may be a compound of formula Va: 5 In some embodiments, the compound of formula VII may be a compound of formula VIIi or VIIii:10 In some embodiments, the compound of formula VII may be a compound of formula 15 VIIa: 126210PCT1 - 22 - In some embodiments, the compound of formula VII may be a compound of formula VIIai or VIIaii: 5 In some embodiments, the compound of formula IX may be a compound of formula 10 IXa: R1may be H or an optionally substituted C1-12 alkyl, an optionally substituted C2-12 15 alkenyl or an optionally substituted C2-12 alkynyl. More preferably, R1is H or an optionally substituted C1-6alkyl, an optionally substituted C2-6alkenyl or an optionally substituted C2-6alkynyl. R1may be H or a C1-3alkyl. In some embodiments, R1is H or methyl. 20 In the compound of formula II, III, VI, VII, VIII or IX R1may be H or a C1-3alkyl. More preferably, R1is H or methyl and most preferably R1is methyl. 126210PCT1 - 23 - In the compound of formula IV, V or X R1may be H or a C1-3alkyl. More preferably, R1is H or methyl and most preferably R1is H. R2may be H, an optionally substituted C1-12 alkyl, an optionally substituted C2-12 alkenyl 5 or an optionally substituted C2-12alkynyl. More preferably, R2is an optionally substituted C1-10 alkyl, an optionally substituted C2-10 alkenyl or an optionally substituted C2-10 alkynyl. Most preferably, R2is an optionally substituted C1-8 alkyl, an optionally substituted C2-8 alkenyl or an optionally substituted C2-8 alkynyl. 10 In embodiments where R2is an optionally substituted alkyl, an optionally substituted alkenyl or an optionally substituted alkynyl, the alkyl, alkenyl or alkynyl may be unsubstituted or substituted with one or more of OH, oxo, NH2, optionally substituted C3-12cycloalkyl, optionally substituted C3-12cycloalkenyl, optionally substituted 3 to 8 membered heterocycle, optionally substituted C6-12 aryl and / or optionally substituted 5 15 to 10 membered heteroaryl. More preferably, the alkyl, alkenyl or alkynyl is unsubstituted or substituted with one or more of OH, oxo, NH2, optionally substituted C3-6cycloalkyl, optionally substituted C3-6cycloalkenyl, optionally substituted 5 or 6 membered heterocycle, optionally substituted phenyl and / or optionally substituted 5 or 6 membered heteroaryl. The cycloalkyl, cycloalkenyl and / or heterocycle may be 20 unsubstituted or substituted with one or more of OH, oxo or NH2. The phenyl and / or heteroaryl may be unsubstituted or substituted with one or more of OH or NH2. In some embodiments, R2is H, methyl, ethyl, propyl or butyl. In some embodiment, R2is methyl, propyl or butyl. Alternatively, R2may be a substituted alkyl. 25 Accordingly, R2may be H, methyl, n-propyl, . In the compound of formula II, III, IV, VI, VII or IX R2may be H or a C1-3 alkyl. More preferably, R2is H or methyl and most preferably R2is methyl. 30 126210PCT1 - 24 - In the compound of formula V R2may be H or a C1-3alkyl. More preferably, R2is methyl, ethyl or propyl and most preferably R2is methyl or propyl. In the compound of formula VIII, R2may be an optionally substituted C1-12 alkyl. More 5 preferably, R2is a substituted C1-8 alkyl, and most preferably i . In the compound of formula X, R2may be H or a C1-6alkyl. More preferably, R2is a C3-6alkyl and most preferably . 10 It may be appreciated that in the compound of formula I if L3is present and R7is a sugar moiety then R3may be an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12cycloalkenyl or COOH. More preferably, R3is an optionally substituted C1-12alkyl, an optionally substituted C2-15 12 alkenyl, an optionally substituted C2-12 alkynyl, an optionally substituted C3-9 cycloalkyl, an optionally substituted C3-9 cycloalkenyl or COOH. More preferably, R3is a C1-6alkyl or COOH. More preferably, R3is a C1-3alkyl or COOH, and is most preferably methyl or COOH.20It is noted that a compound of formula II, III, VI, VII or IX is a compound where L3is present. Accordingly, in these compounds, R7may be a sugar moiety and R3may be as defined above. Alternatively, R3may be COOR5, CONR5R5a, CONR5NR5aR5b, CONR5OR5a, 25 CONR5NR5aOR5bor CONR5OOR5a. R3may be COOR5, CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor CONR5OOR5ain any compound of formula I. 126210PCT1 - 25 - It is noted that in formula IV, V, VIII or X, L3is not present. Accordingly, in these compounds R3is COOR5, CONR5R5a, CONR5NR5aR5bor CONR5OR5a, where COOR5is not COOH. 5 R5, R5aand R5bmay each be H, an optionally substituted C1-6alkyl or phenyl. More preferably, R5, R5aand R5bare each H or an optionally substituted C1-3 alkyl. In some embodiments, R5, R5aand R5bmay each be H, a C1-6 alkyl or phenyl. In some embodiments, R5, R5aand R5bare each H or a C1-3 alkyl. 10 In embodiments where one or more of R5, R5aand R5bis an optionally substituted alkyl, the alkyl may be unsubstituted or substituted with one or more of halo, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12aryl, an optionally substituted 5 to 10 membered heteroaryl, oxo, -O-, hydroxy, a sugar moiety, 15 OR9, COOR9, NR9R10, CN, N3, NO2, SR9, SOR9and SO2R9. R9and R10may be as defined above. In some embodiments, where one or more of R5, R5aand R5bis an optionally substituted alkyl, the alkyl may be unsubstituted or substituted with one or more of halo, oxo, -O-, OR9, COOR9, NR9R10, CN, N3, NO2, SR9, SOR9and SO2R9. In some embodiments, where one or more of R5, R5aand R5bis an optionally substituted alkyl, 20 the alkyl may be unsubstituted or substituted with one or more of oxo, OR9, NR9R10, and SR9. In some embodiments, where one or more of R5, R5aand R5bis an optionally substituted alkyl, the alkyl may be unsubstituted or substituted with OR9. R9and R10may be H, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an 25 optionally substituted C3-12cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl. In some embodiments, R9and R10are independently H, a C1-6 alkyl, a C2-6alkenyl or a C2-6alkynyl. In some embodiments, R9and R10are independently H, a C1-3alkyl, a C2-3alkenyl or a C2-3alkynyl. In some embodiments, R930 and R10are both H. Accordingly, in some embodiments, where one or more of R5, R5aand R5bis an optionally substituted alkyl, the alkyl may be unsubstituted or substituted with OH. In some embodiments, R5, R5aand R5bare each H or a C1-3 alkyl optionally substituted 35 with one or more of oxo, OH, NH2 or SH. In some embodiments, R5, R5aand R5bare each H or a C1-3alkyl optionally substituted with one or more OH. In some 126210PCT1 - 26 - embodiments, the alkyl is substituted with one, two or three OH groups. In some embodiments, R5, R5aand R5bare each H or CH(CH2OH)2. In some embodiments, R5, R5aand R5bare each H. 5 Accordingly, in some embodiments, R3may be COOH, CONH2, CONHNH2, CONHOH or CONHCH(CH2OH)2. In some embodiments, R3may be CONH2, CONHNH2 or CONHOH. R4may be a monosaccharide, a disaccharide, a polysaccharide or a derivative thereof. 10 R4may be , wherein X1is a bond or CR18R19; R15to R23are each independently H, OR24, COR24, COOR24, NR24R25, N+R24R25R26, 15 NR24COR25, NR24COOR25, NR24CONR25R26, optionally substituted C1-6alkyl, optionally substituted C2-6alkenyl, optionally substituted C2-6alkynyl, halo and R24to R26are independently H, an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally 20 substituted C3-12cycloalkyl, an optionally substituted C3-12cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12aryl or an optionally substituted 5 to 10 membered heteroaryl. R15may be H 25 R16may be H, OH or NHCOCH3. R17may be OH or H. 30 In embodiments where it is present, R18may be H, CH3 or OCH3. 126210PCT1 - 27 - In embodiments where it is present, R19may be OH, NH2, N(CH3)2 or H. R20may be H. 5 R21may be R22may be H. 10 R23may be OH, CH2OH or CH3. Accordingly, R4may be glucose, galactose, mannose, fucose, fructose, rhamnose, digitoxose, mycarose, 4-O-methyldigitoxose, ribose, 2-N-acetyl-galactosamine, glucuronic acid, N-acettyl-neuraminic acid, ristosamine, 3-N-methyl-4-O-methyl- 15 ristosamine, fucofuranose, rhamnose, nogalose, streptose, furanose, pyranose, olivose, aryl-C-olivose, noviose, 4-O-methyl-noviose, allose, chalcose, chalcomycin, actinospectose, amicetose, mycinose, chalose, mycarose, cladinose, oleandrose, oliose, olivomose, digitalose, olivomycose, evalose, xylose, pentose, quinovose, arcanose, rhodinose, cinerulose, aculose, lyxose, gulose, colitose, mycaminose, cladinose,20 desosamine, megosamine, rhodosamine, duanosamine, nogalamine, 2-deoxy- nogalamine, forosamine, daunosamine, acosamine, eremosamine, angolosamine, vicenisamine, dimethyl-eremosamine, vancosamine, mycosamine or a derivative thereof. In some embodiments, R4is glucose, galactose, mannose, fucose, fructose, rhamnose, digitoxose, mycarose, 4-O-methyldigitoxose, forosamine, ossamine, 25 desosamine, mycosamine or a derivative thereof. R4may some embodiment, R4may . R6may be H or an optionally substituted C1-12 alkyl, an optionally substituted C2-12 30 alkenyl or an optionally substituted C2-12alkynyl. More preferably, R6is H or an 126210PCT1 - 28 - optionally substituted C1-6alkyl, an optionally substituted C2-6alkenyl or an optionally substituted C2-6 alkynyl. R6may be H or a C1-3 alkyl. In some embodiments, R6is H or methyl. Preferably, R6is methyl. 5 R7may be a monosaccharide, a disaccharide, a polysaccaride or a derivative thereof. R7may be , wherein X1is a bond or CR18R19; 10 R15to R23are each independently H, OR24, COR24, COOR24, NR24R25,+R24R25R26, NR24COR25, NR24COOR25, NR24CONR25R26, optionally substituted C1-6alkyl, optionally substituted C2-6alkenyl, optionally substituted C2-6alkynyl, halo and R24to R26are independently H, an optionally substituted C1-16 alkyl, an optionally 15 substituted C2-16 alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12cycloalkenyl, an optionally substituted 3 to 8 membered heterocycle, an optionally substituted C6-12aryl or an optionally substituted 5 to 10 membered heteroaryl. 20 Accordingly, R7may be glucose, galactose, mannose, fucose, fructose, rhamnose, digitoxose, mycarose, 4-O-methyldigitoxose, ribose, 2-N-acetyl-galactosamine, glucuronic acid, N-acettyl-neuraminic acid, ristosamine, 3-N-methyl-4-O-methyl- ristosamine, fucofuranose, rhamnose, nogalose, streptose, furanose, pyranose, olivose, aryl-C-olivose, noviose, 4-O-methyl-noviose, allose, chalcose, chalcomycin, 25 actinospectose, amicetose, mycinose, chalose, mycarose, cladinose, oleandrose, oliose, olivomose, digitalose, olivomycose, evalose, xylose, pentose, quinovose, arcanose, rhodinose, cinerulose, aculose, lyxose, gulose, colitose, mycaminose, cladinose, desosamine, megosamine, rhodosamine, duanosamine, nogalamine, 2-deoxy- nogalamine, forosamine, daunosamine, acosamine, eremosamine, angolosamine, 30 vicenisamine, dimethyl-eremosamine, vancosamine, mycosamine or a derivative 126210PCT1 - 29 - thereof. In some embodiments, R7may be glucose, galactose, mannose, fucose, fructose, rhamnose, digitoxose, mycarose, 4-O-methyldigitoxose, or a derivative thereof. 5 R15may be H. R16may be H, OH or NHCOCH3. R17may be OH or H. 10 In embodiments where it is present, R18may be H, CH3 or OCH3. In embodiments where it is present, R19may be OH, OCH3, NH2, N(CH3)2or H. 15 R20may be H. 20 126210PCT1 - 30 - ,

[0003] 126210PCT1 - 32 - 126210PCT1 - 36 - , , 126210PCT1 - 37 - , 126210PCT1 - 39 - 5 In accordance with a second aspect, there is provided a pharmaceutical composition comprising the polyene of the first aspect, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, and a pharmaceutically10acceptable vehicle. 126210PCT1 - 40 - In accordance with a third aspect, there is provided the polyene of the first aspect, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, or the pharmaceutical composition of the second aspect, for use as a medicament. 5 In particular, the inventors have found that the polyenes described herein may be used to treat an infection or a prion disease. Accordingly, in accordance with a fourth aspect, there is provided the polyene of the 10 first aspect, or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof, or the pharmaceutical composition of the second aspect, for use in treating, preventing or ameliorating an infection or a prion disease. According to a fifth aspect of the invention, there is provided a method of treating, 15 preventing or ameliorating an infection or a prion disease in a subject, the method comprising administering to a subject in need of such treatment, a therapeutically effective amount of the polyene of the first aspect, or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof, or the pharmaceutical composition of the second aspect. 20 The infection may be a microbial infection. The microbial infection may be a fungal infection or a parasitic infection. The fungal infection may be a yeast infection. The fungal infection may be caused by a 25 fungus in the genus Aspergillus, Candida, Coccidioides, Cryptococcus, Histoplasma, Fusarium, Scedosporium or Trichosporon. The fungal infection may be caused by Aspergillus fumigatus, Candida albicans or Candida auris, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Fusarium species, Scedosporium species or Trichosporon species. 30 The parasitic infection may be a protozoan parasitic infection. The parasitic infection may be leishmaniasis, Chagas disease or African sleeping sickness. Preferably, the parasitic infection is leishmaniasis. 35 Alternatively, the infection may be a viral infection. The viral infection may be a HIV infection, Japanese encephalitis virus, rubella virus, Enterovirus 71 (EV71), vesicular 126210PCT1 - 41 - stomatitis virus (VSV), herpes simplex virus type 1 (HSV-1), Sindbis virus, vaccinia virus or a coronaviruses (e.g. SARS-CoV-2). In particular, in embodiments where the infection is a parasitic infection or a viral 5 infection or the compounds are for use in treating a prion disease, the compound may be a compound of formula III. The compound of formula III may be a compound of formula IIIii or IIIaii. It will be appreciated that these compounds have a core similar to AmB, which is reported to be effective against leishmaniasis, HIV and prion disease. 10 The term “preventing” may be understood to mean reducing the likelihood of the patient developing a microbial infection. It will be appreciated that the polyene described herein, or a pharmaceutically acceptable salt or solvate thereof, may be used in a medicament which may be used in a 15 monotherapy (i.e. use of the polyene alone), for treating, ameliorating, or preventing a microbial infection. Alternatively, the polyene may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing a microbial infection. 20 The polyene may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person 25 or animal in need of treatment. The polyene may be combined with lipid based preparations, for instance AmB formulations in development include lipid complex, liposomal and colloidal dispersion, varying in lipid composition, shape and size of the carrier. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given. 30 Medicaments comprising the polyene described herein may be used in a number of ways. Compositions comprising the polyene of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin. 35 126210PCT1 - 42 - The polyene according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly 5 advantageous when long-term treatment with the polyene used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection). The polyene and compositions according to the invention may be administered to a 10 subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion). In a preferred embodiment, the polyene is administered orally. Accordingly, the 15 polyene may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. The oral formulation could include lipid, polyethylene glycol or chitosan based nanoparticles, lecithin based micelles, carbon nanotubes, cubosomes and / or cochleates. 20 It will be appreciated that the amount of the polyene that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the polyene, and whether it is being used as a monotherapy, or in a combined therapy. The frequency of administration will also be influenced by the half-life of the polyene within the subject being treated. 25 Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular polyene in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the microbial infection. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, sex, diet, and time of 30 administration. The polyene may be administered during or after onset of the microbial infection to be treated. Daily doses may be given as a single administration. Alternatively, the polyene may be given two or more times during a day. 35 126210PCT1 - 43 - Generally, a daily dose of between 0.01µg / kg of body weight and 500mg / kg of body weight of the polyene according to the invention may be used for treating, ameliorating, or preventing a microbial infection. More preferably, the daily dose is between 0.01mg / kg of body weight and 400mg / kg of body weight, more preferably between 5 0.1mg / kg and 200mg / kg body weight, and most preferably between approximately 1mg / kg and 100mg / kg body weight. A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. 10 Alternatively, a slow release device may be used to provide optimal doses of the polyene according to the invention to a patient without the need to administer repeated doses. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific 15 formulations comprising the polyene according to the invention and precise therapeutic regimes (such as daily doses of the polyene) and the frequency of administration). The inventors believe that they are the first to describe a pharmaceutical composition for treating a microbial infection, based on the use of the polyene of the invention. 20 A “subject” may be a vertebrate, mammal, or domestic animal. Hence, the polyene, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse, cattle, sheep or poultry), pets (e.g. a dog), or may be used in other veterinary applications. Most preferably, however, the subject is a human being. 25 A “therapeutically effective amount” of the polyene is any amount which, when administered to a subject, is the amount of drug that is needed to treat the microbial infection. 30 For example, the therapeutically effective amount of the polyene used may be from about 0.01 mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg. It is preferred that the amount of the polyene is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg. 126210PCT1 - 44 - A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. 5 In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- 10 disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents (i.e. the polyene) according to the invention. In tablets, the polyene may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets 15 preferably contain up to 99% of the polyene. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like. 20 However, the pharmaceutically acceptable vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The polyene according to the invention may be dissolved or suspended in 25 a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples 30 of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an 35 oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for 126210PCT1 - 45 - pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be 5 utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The polyene may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. 10 The pharmaceutically acceptable vehicle may be or comprise a drug carrier, optionally a nanoparticle drug carrier. The polyene and compositions of the invention may be administered in the form of a 15 sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The polyene used according to the invention can also be administered orally either in liquid or solid composition form. 20 Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions. 25 It may be appreciated that the polyene of the first aspect may be used to effectively treat infections of plant pathogens. In a sixth aspect, there is provided an agrochemical composition comprising the polyene of the first aspect. 30 In a seventh aspect, there is provided use of the polyene of the first aspect or the agrochemical composition of the sixth aspect to treat a disease in a plant or a mushroom. 35 In accordance with an eighth aspect, there is provided a method of treating a disease in a plant or a mushroom, the method comprising contacting a plant or mushroom in 126210PCT1 - 46 - need of such a treatment with the polyene of the first aspect or the agrochemical composition of the sixth aspect. A disease, which may be treated, may be caused by a fungus. The disease may be dry 5 bubble disease of mushrooms, tomato gray mold or strawberry crown rot disease. The fungus may be Lecanicillium fungicola, Botrytis cinerea or Phytophthora cactorum. The compound may be a compound of formula IV. It may be appreciated that these compounds are based on Pim, which is an effective agent against dry bubble disease of 10 mushrooms caused by Lecanicillium fungicola and against Botrytis cinerea, a causative agent for tomato gray mold. Pim is also used as an effective bio-fungicide in the control of strawberry crown rot disease. The agrochemical composition may comprise one or more solvents in which the 15 polyene is mixed. The amount of solvents in the composition may range from 1 wt% to 99 wt%, or from 30 wt% to 80 wt%. Suitable solvents include, for example, a non-polar water-immiscible solvent, or a polar aprotic water miscible organic solvent. Non-polar solvents include, for example substituted or unsubstituted aliphatic or aromatic hydrocarbons and esters of plant oils or mixtures thereof. Non-limiting examples of 20 aromatic hydrocarbons include benzene or substituted benzene derivatives such as toluene, xylene, 1,2,4-trimethylbenzene, naphthalene or mixtures thereof. In one embodiment, a solvent includes a mixture of napthalen and 1,2,4-trimethylbenzene. In another embodiment, a solvent is Aromatic 150, a heavy aromatic naptha solvent containing <10% naphthalene and <1.7% 1,2,4-trimethylbenzene. 25 Alkyl esters can also be used as non-polar, water immiscible solvents. Plant oils may be esterified with various alcohols to form alkyl esters of plant oils. Fatty acids of these plant oils have 5 to 20, or 6 to 15 carbon atoms. Alkyl esters of plant oils include, without limitation, methyl, ethyl and butyl esters of canola (B. napus), linseed, 30 safflower (Carthamus tinctorius L), soybean and sunflower oils. In one embodiment, the solvent is a mixture of methyl esters. A specific non-limiting example of methyl esters is Agent 2416-21 manufactured by Stepan Company (22 W. Frontage Road, Northfield, Illinois). 126210PCT1 - 47 - Water-miscible polar aprotic solvents can include, for example, alkyl lactates, isopropyl lactate, alkyl carbonates, polyethylene glycols, polyethylene glycol alkyl ethers, polypropylene glycols, and polypropylene glycol alkyl ethers, or mixtures thereof. 5 The agrochemical composition may comprise one or more adjuvants. An adjuvant may enhance or improve performance, for example. Adjuvants may be added to the composition at the time of formulation, or by the applicator to a mix prior to treatment. Adjuvants include, for example surfactants (emulsifier), crop oil, fertilizers, dispersing agents, compatibility agents, foaming activators, foam suppressants, correctives, and 10 spray colorants (dyes). An adjuvant may be present in any desired amount. For example, a formulation may contain 1 wt% to 3 wt% adjuvant, 3 wt% to 8 wt% of adjuvant, 8 wt% to 16 wt% adjuvant, 17 wt% to 30 wt% adjuvant, or 30 wt% or (e.g.40 wt% or more) more adjuvant. 15 The agrochemical composition may comprise one or more surfactants. A surfactant may increase solubility of the polyene in a solution. A surfactant may also affect spray retention, droplet spreading, and dry rates. A surfactant may be anionic or non-ionic. Examples of anionic surfactants include phosphoric mono- and di- esters of long-chain alcohols having 14 to 22 carbon atoms and the salts thereof; phosphoric mono-and di- 20 esters of alkylene oxide addition products of long-chain alcohols having 14 to 22 carbon atoms and the salts thereof; alkylsulphates having 14 to 22 carbon atoms; polyoxyethylene alkyl ether sulphates of alcohols having 14 to 22 carbon atoms; alkane sulphonates having 14 to 22 carbon atoms; and olefin sulphonates having 14 to 22 carbon atoms. 25 Suitable non-ionic surfactants include, for example, ethoxylated fatty acids, alcohol ethoxylates, tristyrylphenol ethoxylates, ethoxylated sorbitan fatty acid esters or mixtures thereof. Ethoxylated fatty acids include castor or canola oil ethoxylates having at least 25, preferably 27 to 37 ethoxy units, such as Sunaptol RTM CA350 (castor oil 30 ethoxylate with 35 ethoxy units) of Uniqema (formerly ICI Surfactants), Mergital RTM EL33 (castor oil ethoxylate with 33 ethoxy units) of Henkel KGaA, Eumulgin RTM C03373 (canola oil ethoxylate with 30 ethoxy units) of Henkel KGaA and Ukanil RTM 2507 (castor oil ethoxylate) of Uniqema. 35 Surfactants may be present in any desired amount. For example, a surfactant may be present in an amount of about 0.1 to about 30 wt%. In a particular embodiment, a 126210PCT1 - 48 - surfactant is present in an amount of about 1 to about 9 wt%. In another embodiment, a surfactant is present in an amount of about 10 to about 20 wt%. The agrochemical composition may comprise one or more emulsifiers. An emulsifier is 5 a type of surfactant typically used to keep emulsion well-dispersed. Non-limiting examples of the emulsifier include Agent 2201-76, Agent 2416-20, Emulpon CO-360, T- Det C-40(R), and Agnique(TM) SBO-IO. Agent 2201-76 is manufactured by Stepan Company (22 W. Frontage Road, Northfield, Illinois), which is a blend of nonionic and anionic surfactants (82%). The ingredients in Agent 2201-76 are alkylbenzene sulfonate 10 and fatty acid ethoxylate, aromatic petroleum hydrocarbon, 1-hexanol and naphthalene. Agent 2416-20 is also manufactured by Stepan Company (22 W. Frontage Road, Northfield, Illinois), which is a blend of nonionic and anionic surfactants (35- 37%). Agent 2416-20 also includes aromatic petroleum hydrocarbon (57-58%), and naphthalene (6-7%). Emulpon CO- 360 is manufactured by Akzo Nobel Chemicals Ltd. 15 (525 West Van Buren, Chicago, Illinois), which contains ethoxylated castor oil (100% by weight) and oxirane (<0.001% by weight). T-Det C-40(R) may be purchased from Harcros Organics (5200 Speaker Road., P.O. Box 2930, Kansas City, Kansas), or from Akzo Nobel Chemicals Ltd. (525 West Van Buren, Chicago, Illinois), which is a non- ionic emulsifier, and a brand of ethoxylated (polyethoxylated) castor oil. Agnique(TM) 20 SBO-IO is manufactured by Cognix GmbH headquartered in Monheim, Germany, which contains alkoxylated triglycerides as an ethoxylated soybean oil. A crop oil, or a crop oil concentrate, may be used to increase the efficacy. Although not wishing to be bound by any particular theory, a crop oil is believed to keep the leaf 25 surface moist longer than water, which in turn allows more time for the composition to penetrate, thereby increasing the amount of the composition that will enter the plant. A crop oil can improve uptake of an agrochemical by a plant. A crop oil can therefore improve, enhance, increase or promote efficacy or activity. Crop oils may contained from 1 wt% to 40 wt%, or 1 wt% to 20 wt% in the formulation. A crop oil can be derived 30 from either petroleum oil or vegetable oil. Non-limiting examples of crop oil include soybean oils and petroleum based oils. The agrochemical composition of the invention may be in customary formulations. Non-limiting examples include solutions, emulsions, suspensions, wettable powders, 35 powders, dusts, pastes, soluble powders, granules, pellets, emulsifiable concentrate, oil spray, aerosol, natural and synthetic materials impregnated with active compound, and 126210PCT1 - 49 - very fine capsules (e.g. in polymeric substances). In certain embodiments, the composition is in a form of an emulsifiable concentrate, wettable powder, granule, dust, oil spray or aerosol. 5 The agrochemical composition may optionally include adherent coatings. Such coatings include those that aid the polyene to adhere to the intended environment, for example, a plant being treated. Adherent coatings include carboxymethylcellulose, natural and synthetic polymers in various forms, such as powders, granules or latexes. Other adherent coatings include gum arabic, polyvinyl alcohol and polyvinyl acetate. 10 Phospholipids, such as cephalins and lecithins, and synthetic phospholipids are also examples of adherent coatings. Further additives may be mineral and vegetable oils. Colourants can also be included in the compositions. Non-limiting examples are inorganic pigments, such as iron oxide, titanium oxide and Prussian Blue, and organic 15 dyestuffs, such as alizarin dyestuffs, azo dye-stuffs and metal phthalocyanine dyestuffs, and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. The agrochemical compositions according to the invention can be applied in the form of 20 ready mixes. Agrochemical compositions can also be formulated individually and mixed upon use, i.e. applied in the form of tank mixes. The compositions of the invention can be used as such or in the form of their formulations, and furthermore also as mixtures. The compositions may also be mixed with other active compounds, such as other fungicides, insecticides, acaricides, nematicides, bird repellents, growth substances, 25 plant nutrients and agents which improve soil structure. For particular application purposes, in particular when applied post-emergence, formulations such as mineral or vegetable oils which are tolerated by plants (for example the commercial product "Oleo DuPont 1 IE") or ammonium salts such as, for example, ammonium sulphate or ammonium thiocyanate, as further additives can be included. 30 The compositions can be used as such, in the form of their formulations or in the forms prepared therefrom by dilution of a concentrated form, such as ready-to-use or concentrated liquids, solutions, suspensions, emulsions, or solids, such as, powders, pastes, granules and pellets. They are dispersed in the customary manner, for example 35 by watering, spraying, atomizing, dusting or scattering. 126210PCT1 - 50 - The agrochemical compositions of the invention can be produced by mixing or suspending one or more stabilizers, an active ingredient, and optionally an adjuvant, a diluent or a solvent. In certain embodiments, compositions of the invention can be produced, for example by first mixing or suspending one or more polyenes with a 5 diluent or solvent. Next, the appropriate amount of adjuvant is combined to the resulting mixture containing the polyene. The polyene can be added at the end and blended until the formulation becomes mostly or entirely homogeneous. Plants that may be treated with the agrochemical composition are generally referred to 10 herein as “crop plants”. Mushrooms that may be treated with the agrochemical composition are generally referred to herein as “crop mushrooms”. The term “crop plants” and “crop mushrooms” as used herein, includes any edible or non-edible plant or mushroom, including decorative, plant species with commercial value, which are planted and cultivated for commercial use. Thus, crop plants include floral and non- 15 floral plants, trees, vegetable plants, turf, and ground cover. Non-limiting specific examples of crop plants include canola, flax, peas, lentils, beans, linola, mustard, chickpeas, sunflowers, potatoes, seedling alfalfa, onions, soybeans and turf grass. The term "plants" is meant to include germinant seeds, emerging seedlings, and established vegetation, including roots and above-ground portions (for example, leaves, stalks, 20 flowers, fruits, branches, limbs, root, etc.). The term “turf” used herein refers to grass which grow in areas in which they are desired, or purposely planned for and maintained, for example, a lawn. Turf also refers to a sod, where the surface layer of ground consisting of a mat of grass and grass roots. A non-limiting example of a crop mushroom is white button mushroom. 25 The application rate of polyene varies depending, for example, on the crop being treated with the agrochemical composition. In general, the application rate may be from 0.01 kg / ha to 5.00kg / ha or from 0.03 kg / ha to 3.00kg / ha of the polyene. 30 The polyenes of the first aspect could also be used to preserve food. Accordingly, in accordance with a ninth aspect, there is provided a preservative comprising the polyene of the first aspect. 35 The preservative is preferably a foodsafe preservative. 126210PCT1 - 51 - In accordance with a tenth aspect, there is provided a foodstuff, comprising the polyene of the first aspect or the preservative of the ninth aspect. In accordance with an eleventh aspect, there is provided use of the polyene of the first 5 aspect or the food preservative of the ninth aspect to preserve a foodstuff. The compound may be a compound of formula IV. It may be appreciated that these compounds are based on Pim, which is used as preservative in yogurt, mayonnaise, bakery items, cured meat products, fresh juices and carbonated drinks without 10 affecting the organoleptic properties. It is believed that the methods used to produce the polyenes of the first aspect are novel and inventive per se. 15 The present inventors have identified glycosyltransferase enzymes that are capable of glycosylating polyenes at the R7position. It may be appreciated that the R7position may be referred to as the C35 position in some polyenes, or a corresponding position in smaller ring polyenes. 20 Thus, in accordance with a twelfth aspect, there is provided a method of producing a polyene comprising a sugar moiety, the method comprising contacting a polyene with a glycosyltransferase enzyme in the presence of a nucleotide-linked sugar to thereby produce the polyene comprising a sugar moiety. 25 Preferably, the method produces a polyene of the first aspect. Referring to Formula I, the method may add the sugar moiety to a polyene at the R4position and / or the R7position. Accordingly, the polyene produced may comprise a sugar moiety at the R4position and / or at the R7position. 30 In an embodiment, the method preferably adds the sugar moiety to a polyene at the R7position. Accordingly, the polyene produced preferably comprises a sugar moiety at the R7position. 35 Accordingly, the polyene which is contacted with the glycosyltransferase enzyme in the presence of a nucleotide-linked sugar, may be a polyene of formula VIII: 126210PCT1 - 52 - 5 wherein, R1to R4, R6, L1and L2are as defined in relation to the first aspect. Similarly, the polyene which is produced in the method of the twelfth aspect may be a polyene of formula IX: 10 wherein R7ais a sugar moiety. The sugar moiety may be as defined in relation to the first aspect. 15 Alternatively, or additionally, the method may comprise adding the sugar moiety to a polyene at the R4position. Accordingly, the polyene produced may comprise a sugar moiety at the R4position. Accordingly, the polyene which is contacted with the glycosyltransferase enzyme in the 20 presence of a nucleotide-linked sugar, may be a polyene of formula XV: 126210PCT1 - 53 - (XV) wherein, R1to R3and L1to L3are as defined in relation to the first aspect. 5 Similarly, the polyene which is produced in the method of the twelfth aspect may be a polyene of formula XVI: 10 wherein R4is a sugar moiety. The sugar moiety may be as defined in relation to the first aspect. In an embodiment, the method may comprise adding a sugar moiety to a polyene at the 15 R4position and a sugar moiety to the polyene at the R7position. Accordingly, the polyene produced may comprise a sugar moiety at the R4position and a sugar moiety at the R7position. Accordingly, the polyene which is contacted with the glycosyltransferase enzyme in the 20 presence of a nucleotide-linked sugar, may be a polyene of formula XVa: Similarly, the polyene which is produced in the method of the twelfth aspect may be a 25 polyene of formula XVIa: 126210PCT1 - 54 - wherein R7ais a sugar moiety. 5 The method may comprise: - contacting a polyene with a first glycosyltransferase enzyme in the presence of a first nucleotide-linked sugar to thereby produce a polyene comprising a first sugar moiety; and - subsequently contacting the polyene comprising the first sugar moiety with a 10 second glycosyltransferase enzyme in the presence of a second nucleotide-linked sugar to thereby produce a polyene comprising first and second sugar moieties. The polyene comprising first and second sugar moieties may comprise sugar moieties at the R4and R7positions. 15 In an embodiment, contacting the polyene with the first glycosyltransferase enzyme in the presence of the first nucleotide-linked sugar produces a polyene comprising a first sugar moiety at the R4position. In an embodiment, contacting the polyene comprising the first sugar moiety at the R4position with a second glycosyltransferase enzyme in the 20 presence of a second nucleotide-linked sugar provides a polyene comprising first and second sugar moieties at the R4and R7positions. In an alternative embodiment, contacting the polyene with the first glycosyltransferase enzyme in the presence of the first nucleotide-linked sugar produces a polyene 25 comprising a first sugar moiety at the R7position. In an embodiment, contacting the polyene comprising the first sugar moiety at the R7position with a second glycosyltransferase enzyme in the presence of a second nucleotide-linked sugar provides a polyene comprising first and second sugar moieties at the R7and R4positions. 30 126210PCT1 - 55 - Preferably, the polyene produced by the method of the twelfth aspect is not . 5 In some embodiments, the method comprises the addition of a further sugar moiety to a polyene, wherein the polyene comprises a first sugar moiety and the further sugar moiety is added at the R7position. The first sugar moiety may be at the R4position. Thus, the method comprises contacting a polyene comprising a first sugar moiety with 10 a glycosyltransferase enzyme in the presence of a nucleotide-linked sugar to thereby produce a polyene comprising at least first and second sugar moieties, wherein the second sugar moiety is present at the R7position. The inventors have discovered that the polyenes of the first aspect may be produced15using glycosyltransferase enzymes identified in Streptomyces noursei, Streptomyces netropsis, Streptomyces albulus, and Streptomyces kasugaensis. Specifically, the inventors have discovered that these glycosyltransferase enzymes are capable of glycosylating polyenes at the R7position. 20 A glycosyltransferase from S. noursei that may be used in the method of the twelfth aspect to add a sugar moiety to a polyene at the R7position is referred to as NysSV, and 126210PCT1 - 56 - has the amino acid sequence identified under GenBank accession number ANZ14691.1, and set out in SEQ ID NO: 1: MRVLLITSPSATHFLPMVPLAWALRAAGHDVLVAGQPDVLDAVAGAGLNAVPTGRRAGIDDAMRELLFRP GLRPFECVGRWTPEMLAAFPPVWQRHSADVLPRYLDLARAHRPDLIVCDVMEFNAPVVGAQLGIPVVRHR 5 YGVDPLLGGVGPAARAALRSLHEELGLAELPEPTAVIDPCPADLQLPGVAPGLAMRYVPYNGNGVLPDWL RAERRAGAPPRRVLVSLGSHTLALNGVPLLRGILAAAGDGSSGVEVLATVPEAYRAELGPVPDTVRLIDP LPLHLIAGDCAAVVHHGGAGTGMTVAGLGVPQLVLPQFADTFAFAERLAAVGAGVAVDTVEQQNDPRFLR KALGTLLGEPSYGHAAERLRHTMEAMPPPAALVPELARIAAGAGA [SEQ ID No: 1] 10 A glycosyltransferase from S. netropsis that may be used in the method of the twelfth aspect to add a sugar moiety to a polyene at the R7position is referred to as MycS3, and may have one of the amino acid sequences set out in SEQ ID Nos: 2-4. 15 MycS3 may have the amino acid sequence set out in SEQ ID NO: 2, (and may be referred to as MycS3a): MRVLIIPSPVVTHLMPLVPLAWALRAAGHELLVVGQPDVMGVARQAGLNAVSIGDRFGMEDVFHSMLVPG KRPIELWGRLDPAHLEHFPPVWKDHSDRVLPAYLELARAYRPDLIVADPMEFNSLVVGGLLGVPVLHHRF GVDAVSEPVRAAARIALRDSCRALGLDELPDPGIQLDPCPPDLQLPGLLQARPIRYVPFNGSGEVPSWLR 20 AERPSAPGKRRVVVSLGTRTLALHGVPFMRGLLRAFEGLRDVEAIATVPGAFRDEIGAVPGNVRMTDPVP LHLLVETCDAVVHHGGSGTVLTTVHAGLPHLVLPQMADQFGHADQLVATGAGIMIDDAAGQNDPVCLRGA LEELLTDPGYAKGAWELREAMREMPAPSEVVAGLGRLL [SEQ ID NO: 2] 25 MycS3 may have the amino acid sequence set out in SEQ ID NO: 3, (and may be referred to as MycS3b): VTTVDRAAPLRVLIIPSPVVTHLMPLVPLAWALRAAGHELLVVGQPDVMGVARQAGLNAVSIGDRFGMED VFQSMLVPGKRPIELWGRLDPAHLAHFPPVWKDHSDRVLPAYLELARAYRPDLIVADPMEFNSLVVGGLL GVPVLHHRFGVDAVSEPVRAAARIALRDSCRALGLDELPDPGIQLDPCPPDLQLPGLLKALPIRYVPFNG 30 SGEVPSWLRAERPSAPGKRRVVVSLGTRTLVLNGVPFMRDLLRAFEGLRDVEAIATVPEAFRDEIGAVPG NVRMTDPVPLHLLVETCDAVVHHGGSGTVLTTVHAGLPHLVLPQMADQFGHADQLVATGAGIMIDDAAGQ NDPVCLRGALEELLCDPGYAKGAWELREAMREMPAPSEVVAGLGRLL [SEQ ID NO: 3] 35 MycS3 may have the amino acid sequence identified under NCBI accession number WP 242626303.1 (DUF1205 domain-containing protein [Streptomyces netropsis]), and set out in SEQ ID NO: 4, (and may be referred to as MycS3c): 126210PCT1 - 57 - MSTVDRGAPLRVLIIPSPVVTHLMPLVPLAWALRAAGHELLVVGQPDVMGVARQAGLNAVSIGDRFGMED VFHSMLVPGKRPIELWGRLDPAHLEHFPPVWKDHSDRVLPAYLELARAYRPDLIVADPMEFNSLVVGGLL GVPVLHHRFGVDAVSEPVRAAARIALRDSCRALGLDELPDPGIQLDPCPPDLQLPGLLQARPIRYVPFNG SGEVPSWLRAERPSAPGKRRVVVSLGTRTLALHGVPFMRGLLRAFEGLRDVEAIATVPGAFRDEIGAVPG 5 NVRMTDPVPLHLLVETCDAVVHHGGSGTVLTTVHAGLPHLVLPQMADQFGHADQLVATGAGIMIDDAAGQ NDPVCLRGALEELLTDPGYAKGAWELREAMREMPAPSEVVAGLGRLL [SEQ ID NO: 4] A glycosyltransferase from S. kasugaensis that may be used in the method of the 10 twelfth aspect to add a sugar moiety to a polyene at the R7position is referred to as KfuSV, and has the amino acid sequence identified under NCBI accession number WP_094790982.1, and set out in SEQ ID NO: 5: MRVLMIPSPAATHFMPLVPLAWALRAAGHELLVAGQPDVLGVARQAGLNAVSLGDWFRADEELRRLLPPG KRPLEVIGRWTAEQLSEFPPNWLVHSERVLPEYVAFAREFRPDVLVSDALECNVLAVGGALGIPVVHHRY 15 GVDPLSGPFLAGARRALRRHCAELGLAELPDPTVVLDPCPPGLQLPGLAPGRPIRYVPFNGSGELPGWLR ERRPAGSPVRRVAVSLGSMTLELNGVPLLRHILAAFEGLPDVEAVATVPEVHRAAVGPVPDGVRMVDPVP LHLLFADCDAVVHHGGTGTGMTATSFGLPQLVLPQLADQFAYGEQLAAAGAAVILDGAAHQDDPVRLRAA LESVVTEPGHRKAAEELRRAMGEMPAPSRVAADLEQLVQVRDRVR [SEQ ID NO: 5] 20 A glycosyltransferase from S. albulus that may be used in the method of the twelfth aspect to add a sugar moiety to a polyene at the R7position is referred to as NysSV1056. Having discovered that S. albulus is capable of producing diglycosylated nystatin, the 25 inventors sought to investigate the enzyme responsible for producing the diglycosylated polyenes. The inventors discovered that S. albulus contains a NysSV homolog glycosyltransferase that is capable of glycosylating polyenes at the R7position. This enzyme, NysSV1056, was found to have the same level of glycosylation activity as NysSV when tested on Nys and AmB under similar conditions. 30 NysSV1056 has the amino acid sequence set out in SEQ ID NO: 6: MTPARRGPAATPEARMRVLLITSPSATHFLPMVPLTWALRAAGHDVLVAGQPDVLDAVAGAGLNAVPTGR RAGIDDAMRELLFRPGLRPFECVGRWTPEMLAAFPPVWQRHSADVLPRYLDLARAHRPDLIVCDVMEFNA PVVGAQLGIPVVRHRYGVDPLLGGVGPAARAALRSLHEELGLAELPEPTAVIDPCPADLQLPGVAPGLPM 35 RYVPYNGNGVLPDWLRAERRAGVSPRRVLVSLGSHTLALNGVPLLRGILAAAGDGPTGVEVLATVPEAYR AELGPVPDTVRLIDPLPLHLIAGDCAAVVHHGGAGTGMTVAALGVPQLVLPQFADTFAFAERLAAVGAGV AVDTVEQQNDPRFLRKALGTLLGEPSYGHAAERLRHTMEAMPPPAALVPELARIAAGAGA 126210PCT1 - 58 - [SEQ ID NO: 6] The inventors have also discovered that the polyenes of the first aspect may be produced using glycosyltransferase enzymes identified in Streptomyces netropsis and 5 Streptomyces kasugaensis. Specifically, the inventors have discovered that these glycosyltransferase enzymes are capable of glycosylating polyenes at the R4position. A glycosyltransferase from S. netropsis that may be used in the method of the twelfth aspect to add a sugar moiety to a polyene at the R4position is referred to as MycDI, and 10 has the amino acid sequence identified set out in SEQ ID No: 30: MGSTRRPILFVSYPESGLLNPLLVLAEELSRRGVEDLWFATDENRRSEIEALKVDTPVEFASLGGVVPEM SAVTWDDKTYREVTQRSRFKAHRAVIKHSFAPRSRVAKYRALEAVVDEVKPALMVIESMCQYAYELAVTK KIPFVLGVPFLPSNVLTSHVPFAKSYTPAHFPVPHTGLPLDMTPVQRAANQLFRLRTLGLFLTSDMRKVV EEDNQVREELGIAPEARGMMVRIDKSELVLCYSVPELDYPFPVPPKMRLVGTMVPPLPQAPEDDGLSDWL 15 AHNDSVVYMGFGTITRLTRAHVASLVEVARRMSDRHQFLWKLPKEQQKLLPPAELLPDNLRIVDWVPSQL DVLAHPSVKVFFTHAGGNGFHEGLYFGKPLVVRPLWVDCDDQARRGQDFGVSLKLDRPETVDTDDVVDKL ARVIGDRSFRERAEHFGQILRAAGGRGAAADLILGLPALAKD [SEQ ID No: 30] 20 A glycosyltransferase from S. kasugaensis that may be used in the method of the twelfth aspect to add a sugar moiety to a polyene at the R4position is referred to as KfuDI, and has the amino acid sequence identified set out in SEQ ID No: 72: MESIRRPILFVSYAESGLLNPLLVLAGELSRRGVEDLWFATDENRRADIKALNAGTPVEFASLGGTVSEM SAVTWDDETYRQVTQRSRFKAHRAVIRHSFAPRSRVEKYRALAAVVAEVRPALMVIESMCQFGYELAITE 25 GIPFVLGVPFVPSNVLTSHVPFAKSYTPPGFPVPHSGLPGDMNFAQRMTNQLFKWRTMGMFTAPAMRKKV AEDVAVRTELGIAPEARGMMARIDAAEMVLCYSVPELDYPFPVPGSMRLVGPMVPPLPQAPDDDGLSEWL DDQDSVIYMGFGTITRLTREQVAALIEVTRRLADRHQVLWKLPTEQQELLPPRETLPGNLRIESWVPSQL DVLGHPSVKVFFTHAGGNGFHEGLYFGKPLVVRPLWVDCDDQAVRGQDFGVSLTLDRPETLDADDVTDKL TRVLDDPAFRERAEHFATLLRAAGGRSTAADLLLGLPALAAN 30 [SEQ ID No: 72] A glycosyltransferase from Streptomyces nodosus that may be used in the method of the twelfth aspect to add a sugar moiety to a polyene at the R4position is referred to as AmphDI, and has the amino acid sequence identified set out in SEQ ID No: 73: 35 MGAHRRPILFVSYAESGLLNPLLVLAEELSRRGVEDLWFATDEKARDQIESASADSELQFASLGDTVSQM SAVTWDDETYAEVTQRSRFKAHRAVIRHSFAPETRVEKYRALEKAVEEIQPALMVIESMCQFGYELAITK GIPFVLGVPFLPSNVLTSHVPFAKSYTPSGFPVPHSGLPGKMSLAQRVENELFRVRTLGMFMTKEIREIV 126210PCT1 - 59 - EEDNRVRGELGISPEARQMMARIDHAEQVLCYSVAELDYPFPMHEKVRLVGTLVPPLPQAPDDEGLSDWL TEQKSVVFMGFGTITRLTREQVASLVEVARRLEGEGHQVLWKLPSEQQHLLPPAEELPANLRIESWVPSQ LDVLAHPNVKVFFTHAGGNGYHEGLYFGKPLVVRPLWVDCDDQAVRGQDFGVSLTVDRPETVDTDDVLDK ITRVLNESSFTERAEYYAGLLKAAGGRTAAADLLLGLPVLAND5 [SEQ ID No: 73] The method may comprise the use of a glycosyltransferase enzyme to add a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 30% sequence identity or at least 40% sequence identity to any one of SEQ ID Nos: 1-6. 10 Alternatively, or additionally, the method may comprise the use of a glycosyltransferase enzyme to add a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 30% sequence identity or at least 40% sequence identity to any one of SEQ ID Nos: 30, 72 and 73. 15 The NysSv, MycS3, KfuSV, and NysSV1056 glycosyltransferase enzymes, having the sequences disclosed as SEQ ID Nos: 1 and 4-6, have been found to share at least 50% sequence identity. A percentage sequence identity matrix is shown below: 20 1: NysSV 100.00 98.23 54.90 59.03 2: NysSV1056 98.23 100.00 53.90 58.78 3: MycS3 54.90 53.90 100.00 62.89 4: KfuSV 59.03 58.78 62.89 100.00 25 KfuDI and MycDI glycosyltransferase enzymes, having the sequences disclosed as SEQ ID Nos: 30 and 72, have been shown to have around 75% sequence identity to AmphDI having SEQ ID No: 73. Thus, the method may comprise the use of a glycosyltransferase enzyme to add a sugar 30 moiety, wherein the glycosyltransferase enzyme comprises at least 50% sequence identity to any one of SEQ ID Nos: 1-6. Preferably, the glycosyltransferase enzyme comprises at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to any one of SEQ ID Nos: 1-6.35The method may comprise the use of a glycosyltransferase enzyme to add a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 30% sequence 126210PCT1 - 60 - identity, at least 40% sequence identity or at least 50% sequence identity to all of SEQ ID Nos: 1-6. Preferably, the glycosyltransferase enzyme comprises at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to all of SEQ ID Nos: 1-6. 5 Alternatively, or additionally, the method may comprise the use of a glycosyltransferase enzyme to add a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 50% sequence identity to any one of SEQ ID Nos: 30, 72 and 73. Preferably, the glycosyltransferase enzyme comprises at least 55%, at least 60%, at least 70%, at least 10 80%, or at least 90% sequence identity to any one of SEQ ID Nos: 30, 72 and 73. The method may comprise the use of a glycosyltransferase enzyme to add a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 30% sequence identity, at least 40% sequence identity or at least 50% sequence identity to all of SEQ 15 ID Nos: 30, 72 and 73. Preferably, the glycosyltransferase enzyme comprises at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to all of SEQ ID Nos: 30, 72 and 73. For the purposes of the present specification, the terms “sequence identity”, “sequence 20 homology”, “sequence similarity”, and similar terms, are to be interpreted as referring to the degree of similarity or relatedness between two sequences, such as two nucleic acid sequences or two protein / amino acid sequences. Thus, for example, if the use of the word “homology” is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two 25 sequences, but rather is looking at the similarity or relatedness between the sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not. 30 The terms “% sequence identity”, “% identical”, “% identity”, “% homology”, “% similarity” and similar terms, are to be understood to refer to the percentage of residues that two or more sequences contain that are the same in corresponding positions when compared and aligned for maximum correspondence. A specified percentage of 35 nucleotides can be referred to as having, for example, at least 70%, 80%, 85%, 90%, 95%, 99% sequence identity or homology over a specified region when compared and 126210PCT1 - 61 - aligned for maximum correspondence. Unless otherwise stated, the determination of % sequence identity / homology is based on a comparison of the entire sequences in question, and not on selected portions. 5 The skilled person will understand that various means for comparing sequences are available. For example, one non-limiting example of a computer sequence alignment program which may be used for determining the percent sequence identity / homology between sequences is the Basic Local Alignment Search Tool (BLAST). 10 In order to identify conserved residues, a blast search was performed against each of the NysSv, MycS3, KfuSV, and NysSV1056 glycosyltransferase enzymes. For each enzyme, 95-100 homologs were identified. Based on the sequences of all of these enzymes, conserved residues and motifs within the sequences were identified. 15 A sequence alignment of the NysSv, MycS3, KfuSV, and NysSV1056 enzymes (i.e. SEQ ID Nos: 1 and 4-6) is shown below:20 . . ... NysSV AGLNAVPTGRRAGIDDAMRELLFRPGLRPFECVGRWTPEMLAAFPPVWQRHSADVLPRYL 10525NysSV1056 AGLNAVPTGRRAGIDDAMRELLFRPGLRPFECVGRWTPEMLAAFPPVWQRHSADVLPRYL 120 MycS3 AGLNAVSIGDRFGMEDVFHSM-LVPGKRPIELWGRLDPAHLEHFPPVWKDHSDRVLPAYL 113 KfuSV AGLNAVSLGDWFRADEELRRL-LPPGKRPLEVIGRWTAEQLSEFPPNWLVHSERVLPEYV 104 *X**** * :: :: : : ** **:* ** * *** * ** *** *:30NysSV DLARAHRPDLIVCDVMEFNAPVVGAQLGIPVVRHRYGVDPLLGGVGPAARAALRSLHEEL 165 NysSV1056 DLARAHRPDLIVCDVMEFNAPVVGAQLGIPVVRHRYGVDPLLGGVGPAARAALRSLHEEL 180 MycS3 ELARAYRPDLIVADPMEFNSLVVGGLLGVPVLHHRFGVDAVSEPVRAAARIALRDSCRAL 173 KfuSV AFAREFRPDVLVSDALECNVLAVGGALGIPVVHHRYGVDPLSGPFLAGARRALRRHCAEL 164 :** .*X*::*.* :* * .**. **:**::**:*** : . .** *** * 3540 . . . . . . NysSV SLGSHTLALNGVPLLRGILAAAGDGSSGVEVLATVPEAYRAELGPVPDTVRLIDPLPLHL 285 NysSV1056 SLGSHTLALNGVPLLRGILAAAGDGPTGVEVLATVPEAYRAELGPVPDTVRLIDPLPLHL 300 126210PCT1 - 62 - MycS3 SLGTRTLALHGVPFMRGLLRAF-EGLRDVEAIATVPGAFRDEIGAVPGNVRMTDPVPLHL 292 KfuSV SLGSMTLELNGVPLLRHILAAF-EGLPDVEAVATVPEVHRAAVGPVPDGVRMVDPVPLHL 283 5 .. ... .. . . 10 NysSV PRFLRKALGTLLGEPSYGHAAERLRHTMEAMPPPAALVPELARIAAGAGA--395 NysSV1056 PRFLRKALGTLLGEPSYGHAAERLRHTMEAMPPPAALVPELARIAAGAGA--410 MycS3 PVCLRGALEELLTDPGYAKGAWELREAMREMPAPSEVVAGLGRLL-------397 KfuSV PVRLRAALESVVTEPGHRKAAEELRRAMGEMPAPSRVAADLEQLVQVRDRVR395 15 * ** ** :: :*.: :.* .**.:* *X Y: :. Y :: Twenty residues were found to be conserved between substantially all (more than 98%) of the identified homologs. These substantially conserved residues are identified in bold and with an “X” or a “Y” in the bottom line of the sequence alignment above, and 20 correspond to residues (numbering based on the NysSV sequence above): P19, G28, H29, D30, G47, P113, D178, P199, N201, G202, G228, G236, P272, P282, G309, P311, G332, P377, P379, L386. Thus, the method may comprise the use of a glycosyltransferase enzyme comprising at 25 least 15 of the following residues (in positions corresponding to positions numbered based on the NysSV sequence above) P19, G28, H29, D30, G47, P113, D178, P199, N201, G202, G228, G236, P272, P282, G309, P311, G332, P377, P379, L386. In some embodiments, the glycosyltransferase enzyme may comprise at least 16, or at least 17, of these residues. Preferably, the glycosyltransferase enzyme comprises at least 18 of 30 these residues. More preferably, the glycosyltransferase enzyme comprises at least 19 of these residues.Most preferably, the glycosyltransferase enzyme comprises all 20 of these residues. Thirteen residues were found to be conserved between all of the homologs. These 35 conserved residues are identified in bold (and not italic), and with an “X” in the bottom line of the sequence alignment above, and correspond to residues (numbering based on the NysSV sequence above) G28, H29, G47, P113, D178, N201, G202, G228, G236, P272, P311, G332, P377. 126210PCT1 - 63 - Thus, the method may comprise the use of a glycosyltransferase enzyme comprising at least 10 of the following residues (in positions corresponding to positions numbered based on the NysSV sequence above) G28, H29, G47, P113, D178, N201, G202, G228, G236, P272, P311, G332, P377. In some embodiments, the glycosyltransferase enzyme 5 may comprise at least 11 of these residues. Preferably, the glycosyltransferase enzyme comprises at least 12 of these residues. Most preferably, the glycosyltransferase enzyme comprises all 13 of these residues. The inventors have identified three motifs that are conserved between substantially all 10 (greater than 98%) of the glycosyltransferase enzyme homologs that may be used in the disclosed method. These motifs are identified as shaded regions in the sequence alignment above. The first motif, the start of which is located in the region of the sequence corresponding 15 to residue 19 of the NysSV sequence above, has the consensus sequence set out in SEQ ID NO: 7, wherein “X” is A, T, or P, and is preferably P, “x” is any amino acid residue, and “Y” is an acidic residue (E or D): XxxxxxxGHY [SEQ ID NO: 7] 20 The second motif, the start of which is located in the region of the sequence corresponding to residue 199 of the NysSV sequence above, has the consensus sequence set out in SEQ ID NO: 8, wherein “X” is P, N, V, A or H, and is preferably P, and wherein “x” is any amino acid residue: 25 XxNG [SEQ ID NO: 8] The third motif, the start of which is located in the region of the sequence corresponding to residue 309 of the NysSV sequence above, has the consensus 30 sequence set out in SEQ ID NO: 69, wherein “X” is G or A, and is preferably G, and wherein “x” is any amino acid residue: XxP [SEQ ID NO: 69] 35 Thus, the method may comprise the use of a glycosyltransferase enzyme comprising at least one of the first, second, and / or third conserved motifs. Preferably, the 126210PCT1 - 64 - glycosyltransferase enzyme comprises at least two of the first, second, and / or third conserved motifs. More preferably, the glycosyltransferase enzyme comprises all three of the first, second, and third conserved motifs. 5 In some embodiments, the glycosyltransferase enzyme comprises all three of the first, second, and third conserved motifs, wherein in the first motif, X is P, in the second motif, X is P, and / or in the third motif, X is G. Preferably, the glycosyltransferase enzyme comprises all three of the first, second, and third conserved motifs, wherein in the first motif, X is P, in the second motif, X is P, and in the third motif, X is G. 10 The “DUF1205 domain” is a conserved region of unknown function found in several glycosyltransferase enzymes of bacterial origin. Notably, this domain is not present in glycosyltransferase enzymes that add a first sugar to polyenes in the R4position (such as the NysDI, AmphDI, and PimD glycosyltransferase enzymes). The inventors have 15 surprisingly found, however, that the DUF1205 domain is a common feature of glycosyltransferase enzymes that are capable of adding a further sugar moiety to a polyene, wherein the polyene comprises a first sugar moiety and the further sugar moiety is added at the R7position. Thus, glycosyltransferase enzymes for use in the method of the twelfth aspect may comprise the DUF1205 domain. For the avoidance of 20 doubt, the DUF1205 domain is identified in the sequence alignment above using underlining, and corresponds to residues 177-276 of the NysSV sequence above (which corresponds to residues 192-291 of the Nys1056SV sequence, residues 176-274 of the KfuSV sequence, and residues 186-283 of the MycSIII sequence). 25 NysSV1056 has the nucleotide sequence set out in SEQ ID NO: 9: ATGACGCCCGCCCGGCGGGGACCCGCCGCCACTCCGGAGGCCCGTATGCGCGTCCTGCTGATCACCAGTC CGTCGGCCACCCACTTCCTGCCGATGGTGCCGCTCACCTGGGCGCTGCGGGCCGCCGGCCACGACGTACT GGTGGCGGGCCAGCCCGACGTCCTGGACGCGGTGGCCGGCGCCGGCCTGAACGCCGTGCCGACCGGGCGG CGCGCGGGCATCGACGACGCGATGCGCGAGCTGCTGTTCCGGCCCGGACTGCGGCCCTTCGAATGCGTCG 30 GCCGCTGGACCCCGGAGATGCTGGCCGCCTTCCCGCCCGTGTGGCAGCGGCACAGCGCGGACGTGCTGCC GCGCTATCTGGACCTGGCCCGGGCGCACCGCCCCGACCTGATCGTCTGCGACGTGATGGAGTTCAACGCC CCGGTGGTCGGCGCCCAGTTGGGCATCCCGGTGGTGCGGCACCGGTACGGCGTCGATCCGCTGCTGGGCG GCGTCGGCCCGGCCGCGCGGGCCGCCCTGCGGTCGCTGCACGAGGAACTGGGACTGGCGGAACTGCCGGA ACCCACCGCGGTGATCGACCCCTGCCCCGCGGACCTGCAGCTCCCGGGCGTCGCCCCGGGCCTGCCCATG 35 CGGTACGTGCCCTACAACGGCAACGGCGTACTGCCGGACTGGCTGCGGGCCGAGCGGCGGGCCGGGGTGT CACCGCGCCGCGTACTGGTCTCGCTCGGTTCGCACACCCTGGCTCTCAACGGCGTCCCGCTGCTGCGCGG CATCCTCGCGGCGGCCGGCGACGGGCCGACGGGCGTCGAGGTCCTGGCGACCGTGCCGGAGGCGTACCGC 126210PCT1 - 65 - GCGGAGCTCGGCCCGGTGCCGGACACGGTACGGCTGATCGACCCGCTGCCGTTGCACCTGATCGCCGGCG ACTGTGCGGCGGTGGTGCACCACGGCGGGGCCGGCACCGGTATGACCGTGGCCGCTCTCGGCGTACCGCA GCTGGTGCTCCCGCAGTTCGCCGACACCTTCGCGTTCGCCGAGCGGCTGGCGGCGGTCGGCGCCGGGGTG GCCGTCGACACCGTCGAGCAGCAGAACGACCCGCGGTTCCTGCGGAAGGCGCTGGGGACGCTCCTCGGGG 5 AGCCGTCCTACGGGCATGCGGCCGAGCGGCTGCGGCACACCATGGAGGCCATGCCGCCACCGGCCGCCCT GGTCCCGGAGTTGGCGCGGATCGCGGCGGGAGCGGGCGCATGA [SEQ ID NO: 9] The method may comprise the use of a glycosyltransferase enzyme to add a sugar 10 moiety, wherein the glycosyltransferase enzyme is encoded by a nucleotide sequence comprising at least 30% sequence identity, at least 40% sequence identity or at least 50% sequence identity to SEQ ID NO: 9. Preferably, the glycosyltransferase enzyme is encoded by a nucleotide sequence comprising at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 9. The nucleotide 15 sequence may be codon optimised, for improved used in an expression vector. 100 homologs of MycDI, including, KfuDI and AmphDI, were identified and analysed using Multiple Sequence Alignment. The most distantly related homologs still shared 76% sequence identity with MycDI. 20 A sequence alignment of the MycDI, KfuDI and AmphDI enzymes (i.e. SEQ ID Nos: 30, 72 and 73) is shown below: 25 ... AmphDI ASLGDTVSQMSAVTWDDETYAEVTQRSRFKAHRAVIRHSFAPETRVEKYRALEKAVEEIQ 120 MycDI ASLGGVVPEMSAVTWDDKTYREVTQRSRFKAHRAVIKHSFAPRSRVAKYRALEAVVDEVK 120 30 KasDI ASLGGTVSEMSAVTWDDETYRQVTQRSRFKAHRAVIRHSFAPRSRVEKYRALAAVVAEVR 120 ****..* :********:** :**************:*****.:** ***** .* *:: AmphDI PALMVIESMCQFGYELAITKGIPFVLGVPFLPSNVLTSHVPFAKSYTPSGFPVPHSGLPG 180 MycDI PALMVIESMCQYAYELAVTKKIPFVLGVPFLPSNVLTSHVPFAKSYTPAHFPVPHTGLPL 180 35 KasDI PALMVIESMCQFGYELAITEGIPFVLGVPFVPSNVLTSHVPFAKSYTPPGFPVPHSGLPG 180 ***********:.****:*: *********:***************** *****:*** AmphDI KMSLAQRVENELFRVRTLGMFMTKEIREIVEEDNRVRGELGISPEARQMMARIDHAEQVL 240 MycDI DMTPVQRAANQLFRLRTLGLFLTSDMRKVVEEDNQVREELGIAPEARGMMVRIDKSELVL 240 40 KasDI DMNFAQRMTNQLFKWRTMGMFTAPAMRKKVAEDVAVRTELGIAPEARGMMARIDAAEMVL 240 .*. .** *:**: **:*:* : :*: * ** ** ****:**** **.*** :* ** AmphDI CYSVAELDYPFPMHEKVRLVGTLVPPLPQAPDDEGLSDWLTEQKSVVFMGFGTITRLTRE 300 MycDI CYSVPELDYPFPVPPKMRLVGTMVPPLPQAPEDDGLSDWLAHNDSVVYMGFGTITRLTRA 300 45 KasDI CYSVPELDYPFPVPGSMRLVGPMVPPLPQAPDDDGLSEWLDDQDSVIYMGFGTITRLTRE 300 **** *******: .:**** :********:*:***:** .:.**::*********** AmphDI QVASLVEVARRLEGEGHQVLWKLPSEQQHLLPPAEELPANLRIESWVPSQLDVLAHPNVK 360 126210PCT1 - 66 - MycDI HVASLVEVARRMS-DRHQFLWKLPKEQQKLLPPAELLPDNLRIVDWVPSQLDVLAHPSVK 359 KasDI QVAALIEVTRRLA-DRHQVLWKLPTEQQELLPPRETLPGNLRIESWVPSQLDVLGHPSVK 359 :**:*:**:**: : **.*****.***.**** * ** **** .*********.**.** 5 AmphDI VFFTHAGGNGYHEGLYFGKPLVVRPLWVDCDDQAVRGQDFGVSLTVDRPETVDTDDVLDK 420 MycDI VFFTHAGGNGFHEGLYFGKPLVVRPLWVDCDDQARRGQDFGVSLKLDRPETVDTDDVVDK 419 KasDI VFFTHAGGNGFHEGLYFGKPLVVRPLWVDCDDQAVRGQDFGVSLTLDRPETLDADDVTDK 419 **********:*********************** *********.:*****:*:*** ** 10 AmphDI ITRVLNESSFTERAEYYAGLLKAAGGRTAAADLLLGLPVLAND 463 MycDI LARVIGDRSFRERAEHFGQILRAAGGRGAAADLILGLPALAKD 462 KasDI LTRVLDDPAFRERAEHFATLLRAAGGRSTAADLLLGLPALAAN 462 ::**:.: :* ****::. :*:***** :****:****.** :15The glycosyltransferase enzyme may be a mutant or variant of any of the enzymes described herein. For example, the glycosyltransferase may be engineered to provide increased or otherwise improved activity, or to provide an altered selectivity or specificity for different sugar nucleotide and / or polyene substrates. X-ray crystal structures or AlphaFold structural predications may be generated for the 20 glycosyltransferases and used to design modifications to the enzyme. AlphaFold uses well known software 29, 30, 31 which have proved to provide reliable structural predictions that may be used to guide engineering. This involves identifying putative sugar nucleotide and polyene bind sites within the AlphaFold structures. Amino acid residues present in these binding sites may then be subjected to site directed 25 mutagenesis to alter the binding sites and properties of the enzymes. One particularly useful approach for improving the properties of enzymes is the iterative saturation mutagenesis (ISM) method. Also, more random mutagenesis approaches such as the well-known error prone PCR method can be used to create libraries of enzymes variants. The mutant enzyme variants may then be screened (tested) for activity with 30 different polyene and sugar nucleotide depending on the preferred glycosylated polyene product that is required. All of the glycosyltransferase enzymes disclosed herein that have been found by the inventors to be capable of catalyzing the addition of a sugar moiety to polyenes at the R735 position (including KfuSV, NysSV, NysSV1056, MycS3) share the same overall folded protein structure. This is clear, for example, from the structures of KfuSV and NysSV1056 shown in Figure 15. The inventors have alphaFold models of the glycosyltransferase enzymes that have 40 been found to be capable of adding of a sugar moiety to polyenes at the R7position (including KfuSV, NysSV, NysSV1056, MycS3). 126210PCT1 - 67 - KfuSV has been found to comprise a N-terminal “sugar nucleotide” domain covering the region of residues 1-197 and C-terminal “aglycone binding” domain spanning the region of residues 198-395. Docking results suggest that the polyene binds to the surface of the aglycone binding C-domain while nucleotide sugar lies in the 5 interdomain region forming contacts with the N-domain residues. Modelling the folded conformation of the other glycosyltransferase enzymes NysSV, NysSV1056, MycS3 reveals that these enzymes have structures that are very closely related to the KfuSV structure, and suggest that these enzymes also include C-terminal 10 domain to which the polyene binds, and an N-domain comprising residues that interact with the sugar. For NysSV and Nys1056SV, the N-domain spans the region of residue 1- 180 and C-domain from residue 181-395, and for MycS3 residues 1-188 make up the N- domain and 189-397 make the C-domain. 15 In view of these findings, the method may comprise the use of a chimeric glycosyltransferase. Chimeric glycosyltransferases may be produced by combining domains, such as an aglycone binding domain from a first glycosyltransferase enzyme and a sugar nucleotide domain from a second glycosyltransferase enzyme.33, 3420 For example, in some embodiments, the sugar binding domain of KfuSV could be replaced by a sugar binding domain from a different glycosyltransferase. The production and use of chimeric glycosyltransferase enzymes in this way may provide enzymes with altered substrate specificity profiles, for example, thereby providing a method for the addition of alternative sugars to the polyene substrate. 25 Prior to contacting the polyene with the glycosyltransferase enzyme in the presence of the nucleotide-linked sugar to produce the polyene comprising a sugar moiety, the method may comprise: - contacting a polyene comprising a sugar moiety with a glycosyltransferase 30 enzyme to thereby remove the sugar moiety from the polyene. The method may subsequently comprise adding a further sugar moiety to the polyene, as described above. 35 The method may comprise the use of a glycosyltransferase enzyme to remove a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 30% sequence 126210PCT1 - 68 - identity or at least 40% sequence identity to any one of SEQ ID Nos: 1-6, 30, 72 and 73. The method may comprise the use of a glycosyltransferase enzyme to remove a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 50% sequence identity to any one of SEQ ID Nos: 1-6, 30, 72 and 73. Preferably, the 5 glycosyltransferase enzyme comprises at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to any one of SEQ ID Nos: 1-6, 30, 72 and 73. In some embodiments, the method may comprise the use of a glycosyltransferase enzyme to remove a sugar moiety, wherein the glycosyltransferase enzyme comprises at 10 least 30% sequence identity or at least 40% sequence identity to any one of SEQ ID Nos: 30, 72 and 73. The method may comprise the use of a glycosyltransferase enzyme to remove a sugar moiety, wherein the glycosyltransferase enzyme comprises at least 50% sequence identity to any one of SEQ ID Nos: 30, 72 and 73. Preferably, the glycosyltransferase enzyme comprises at least 55%, at least 60%, at least 70%, at least 15 80%, or at least 90% sequence identity to any one of SEQ ID Nos: 30, 72 and 73. The polyene comprising a sugar moiety may be a polyene of formula XVII: 20 wherein R4ais a sugar moiety. R4amay be a different sugar moiety to R4in the polyene of formula XVI. Removing the sugar moiety may provide a polyene of formula XV, as defined above. 25 The polyene comprising a sugar moiety may be contacted with the glycosyltransferase enzyme in the presence of a nucleoside diphosphate (NDP)-sugar hydrolase. It may be appreciated that the NDP-sugar hydrolase may be specific to the sugar which is to be removed, e.g. specific to the sugar in the R4aposition. For instance, if the sugar to be 30 removed is a mannose (e.g. R4ais mannose) then the NDP-sugar hydrolase may be NDP-mannose hydrolase. 126210PCT1 - 69 - The NDP-sugar hydrolase may be a guanosine diphosphate (GDP)-sugar hydrolase. Accordingly, in some embodiments, the NDP-sugar hydrolase is a GDP-mannose hydrolase (GDPMH). 5 A GDPMH that may be used has the amino acid sequence set out in SEQ ID No: 74: MFLRQEDFATVVRSTPLVSLDFIVENSRGEFLLGKRTNRPAQGYWFVPGGRVQKDETLEAAFERLTMAEL GLRLPITAGQFYGVWQHFYDDNFSGTDFTTHYVVLGFRFRVSEEELLLPDEQHDDYRWLTSDALLASDNV HANSRAYFLAEKRTGVPGL 10 [SEQ ID No: 74] The method may comprise the use of an NDP-sugar hydrolase enzyme, wherein the NDP-sugar hydrolase enzyme comprises at least 30% sequence identity or at least 40% sequence identity to SEQ ID No: 74. The method may comprise the use of an NDP- 15 sugar hydrolase enzyme, wherein the NDP-sugar hydrolase enzyme comprises at least 50% sequence identity to SEQ ID No: 74. Preferably, the NDP-sugar hydrolase enzyme comprises at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to any one of SEQ ID No: 74. 20 The method may be an in vitro cell-free method. For example, the nucleotide-linked sugar and the polyene may be incubated with the glycosyltransferase enzyme to produce a polyene comprising a sugar moiety in a biotransformation reaction. Thus, the method may comprise contacting a polyene and a nucleotide-linked sugar 25 with a glycosyltransferase enzyme as defined in accordance with the twelfth aspect to produce a polyene comprising a sugar moiety. Indeed, the glycosyltransferase enzyme that may be used in the method of the twelfth aspect may be any glycosyltransferase enzyme that is capable of adding a sugar moiety 30 to a polyene at the R7position when contacted with a polyene and a nucleotide-linked sugar in an in vitro cell-free method. The method may comprise the use of a recombinant enzyme. Thus, the method may comprise the expression and purification of a recombinant glycosyltransferase enzyme, 35 and the subsequent use of the enzyme to produce a polyene comprising a sugar moiety. Various suitable methods for expressing and purifying an enzyme are known. 126210PCT1 - 70 - In some embodiments, the glycosyltransferase enzyme may be expressed in recombinant form from a suitable cell line or protein expression system. The enzyme may be expressed in E. coli or another host cell, for example, and purified for use in the 5 reaction.36In some embodiments, the recombinant glycosyltransferase enzyme may be encoded in a suitable vector, such as a pET28a vector system under the control of a suitable promoter, such as a T7 promoter. 10 In some embodiments, the recombinant glycosyltransferase enzyme may be concentrated and / or purified. Various suitable methods for concentrating and / or purifying an enzyme are known in the art and may be used in the method of the twelfth aspect. 15 In some embodiments, the method comprises the use of a glycosyltransferase enzyme comprising an affinity tag, wherein the method comprises concentrating and / or purifying the enzyme using the affinity tag. The affinity tag may be a His-tag, for example. 20 Any of the glycosyltransferase enzymes as described in accordance with the twelfth aspect may be adapted to include a His-tag. For example, in some embodiments, the glycosyltransferase from S. noursei, or variant 25 thereof defined above, that may be used in the method of the twelfth aspect to add a sugar moiety may comprise an N-terminal his-tag sequence (referred to as NysSV-His tag) as shown in SEQ ID NO: 10: MGSSHHHHHHSSGLVPRGSHMRVLLITSPSATHFLPMVPLAWALRAAGHDVLVAGQPDVLDAVAGAGLNA VPTGRRAGIDDAMRELLFRPGLRPFECVGRWTPEMLAAFPPVWQRHSADVLPRYLDLARAHRPDLIVCDV 30 MEFNAPVVGAQLGIPVVRHRYGVDPLLGGVGPAARAALRSLHEELGLAELPEPTAVIDPCPADLQLPGVA PGLAMRYVPYNGNGVLPDWLRAERRAGAPPRRVLVSLGSHTLALNGVPLLRGILAAAGDGSSGVEVLATV PEAYRAELGPVPDTVRLIDPLPLHLIAGDCAAVVHHGGAGTGMTVAGLGVPQLVLPQFADTFAFAERLAA VGAGVAVDTVEQQNDPRFLRKALGTLLGEPSYGHAAERLRHTMEAMPPPAALVPELARIAAGAGA [SEQ ID NO: 10] 35 126210PCT1 - 71 - In some embodiments, the glycosyltransferase from S. netropsis, or variant thereof defined above, that may be used in the method of the twelfth aspect to add a sugar moiety may comprise an N-terminal his-tag sequence (and may be referred to as MycS3a-His tag) as shown in SEQ ID NO: 11: 5 MGSSHHHHHHSSGLVPRGSHMRVLIIPSPVVTHLMPLVPLAWALRAAGHELLVVGQPDVMGVARQAGLNA VSIGDRFGMEDVFHSMLVPGKRPIELWGRLDPAHLEHFPPVWKDHSDRVLPAYLELARAYRPDLIVADPM EFNSLVVGGLLGVPVLHHRFGVDAVSEPVRAAARIALRDSCRALGLDELPDPGIQLDPCPPDLQLPGLLQ ARPIRYVPFNGSGEVPSWLRAERPSAPGKRRVVVSLGTRTLALHGVPFMRGLLRAFEGLRDVEAIATVPG AFRDEIGAVPGNVRMTDPVPLHLLVETCDAVVHHGGSGTVLTTVHAGLPHLVLPQMADQFGHADQLVATG 10 AGIMIDDAAGQNDPVCLRGALEELLTDPGYAKGAWELREAMREMPAPSEVVAGLGRLL [SEQ ID NO: 11] In some embodiments, the glycosyltransferase from S. netropsis, or variant thereof defined above, that may be used in the method of the twelfth aspect to add a sugar 15 moiety may comprise an N-terminal his-tag sequence (and may be referred to as MycS3b-His tag) as shown in SEQ ID NO: 70: MGSSHHHHHHSSGLVPRGSHVTTVDRAAPLRVLIIPSPVVTHLMPLVPLAWALRAAGHELLVVGQPDVMG VARQAGLNAVSIGDRFGMEDVFQSMLVPGKRPIELWGRLDPAHLAHFPPVWKDHSDRVLPAYLELARAYR PDLIVADPMEFNSLVVGGLLGVPVLHHRFGVDAVSEPVRAAARIALRDSCRALGLDELPDPGIQLDPCPP 20 DLQLPGLLKALPIRYVPFNGSGEVPSWLRAERPSAPGKRRVVVSLGTRTLVLNGVPFMRDLLRAFEGLRD VEAIATVPEAFRDEIGAVPGNVRMTDPVPLHLLVETCDAVVHHGGSGTVLTTVHAGLPHLVLPQMADQFG HADQLVATGAGIMIDDAAGQNDPVCLRGALEELLCDPGYAKGAWELREAMREMPAPSEVVAGLGRLL [SEQ ID NO: 70] 25 In some embodiments, the glycosyltransferase from S. netropsis, or variant thereof defined above, that may be used in the method of the twelfth aspect to add a sugar moiety may comprise an N-terminal his-tag sequence (and may be referred to as MycS3c-His tag) as shown in SEQ ID NO: 71: MGSSHHHHHHSSGLVPRGSHMSTVDRGAPLRVLIIPSPVVTHLMPLVPLAWALRAAGHELLVVGQPDVMG 30 VARQAGLNAVSIGDRFGMEDVFHSMLVPGKRPIELWGRLDPAHLEHFPPVWKDHSDRVLPAYLELARAYR PDLIVADPMEFNSLVVGGLLGVPVLHHRFGVDAVSEPVRAAARIALRDSCRALGLDELPDPGIQLDPCPP DLQLPGLLQARPIRYVPFNGSGEVPSWLRAERPSAPGKRRVVVSLGTRTLALHGVPFMRGLLRAFEGLRD VEAIATVPGAFRDEIGAVPGNVRMTDPVPLHLLVETCDAVVHHGGSGTVLTTVHAGLPHLVLPQMADQFG HADQLVATGAGIMIDDAAGQNDPVCLRGALEELLTDPGYAKGAWELREAMREMPAPSEVVAGLGRLL 35 [SEQ ID NO: 71] 126210PCT1 - 72 - In some embodiments, the glycosyltransferase from S. kasugaensis, or variant thereof defined above, that may be used in the method of the twelfth aspect to add a sugar moiety may comprise an N-terminal his-tag sequence (referred to as KfuSV-His tag) as shown in SEQ ID NO: 12: 5 MGSSHHHHHHSSGLVPRGSHMRVLMIPSPAATHFMPLVPLAWALRAAGHELLVAGQPDVLGVARQAGLNA VSLGDWFRADEELRRLLPPGKRPLEVIGRWTAEQLSEFPPNWLVHSERVLPEYVAFAREFRPDVLVSDAL ECNVLAVGGALGIPVVHHRYGVDPLSGPFLAGARRALRRHCAELGLAELPDPTVVLDPCPPGLQLPGLAP GRPIRYVPFNGSGELPGWLRERRPAGSPVRRVAVSLGSMTLELNGVPLLRHILAAFEGLPDVEAVATVPE VHRAAVGPVPDGVRMVDPVPLHLLFADCDAVVHHGGTGTGMTATSFGLPQLVLPQLADQFAYGEQLAAAG 10 AAVILDGAAHQDDPVRLRAALESVVTEPGHRKAAEELRRAMGEMPAPSRVAADLEQLVQVRDRVR [SEQ ID NO: 12] In some embodiments, the glycosyltransferase from S. albulus, or variant thereof defined above, that may be used in the method of the twelfth aspect to add a sugar 15 moiety may comprise an N-terminal his-tag sequence (referred to as NysSV1056-His tag) as shown in SEQ ID NO: 13: MGSSHHHHHHSSGLVPRGSHTPARRGPAATPEARMRVLLITSPSATHFLPMVPLTWALRAAGHDVLVAGQ PDVLDAVAGAGLNAVPTGRRAGIDDAMRELLFRPGLRPFECVGRWTPEMLAAFPPVWQRHSADVLPRYLD LARAHRPDLIVCDVMEFNAPVVGAQLGIPVVRHRYGVDPLLGGVGPAARAALRSLHEELGLAELPEPTAV 20 IDPCPADLQLPGVAPGLPMRYVPYNGNGVLPDWLRAERRAGVSPRRVLVSLGSHTLALNGVPLLRGILAA AGDGPTGVEVLATVPEAYRAELGPVPDTVRLIDPLPLHLIAGDCAAVVHHGGAGTGMTVAALGVPQLVLP QFADTFAFAERLAAVGAGVAVDTVEQQNDPRFLRKALGTLLGEPSYGHAAERLRHTMEAMPPPAALVPEL ARIAAGAGA [SEQ ID NO: 13] 25 In addition, or as an alternative, to the use of a purified enzyme, the method may be an in vitro cell-free method comprising the use of a cell lysate comprising the glycosyltransferase enzyme. 30 In some embodiments, the method may be an in vitro cell-free method comprising the use of a crude enzyme extract comprising the glycosyltransferase enzyme. Various approaches are known for preparing recombinant enzymes for use in in vitro cell-free biotransformation reactions, and such methods may be used in the method of 35 the twelfth aspect. Such methods, include, for example, immobilisation of the glycosyltransferase enzyme on a suitable solid support, or encapsulation of the glycosyltransferase enzyme within a porous membrane.37 126210PCT1 - 73 - The sugar-nucleotide substrate for use in embodiments of the twelfth aspect comprising an in vitro cell-free method may be obtained from commercial sources. 5 In some embodiments, sugar nucleotides may be obtained by reverse glycosylation. In this approach a glycosylated natural product is incubated with the cognate glycosyltransferase in the presence of excess of nucleoside diphosphate, so that the sugar is removed from the natural product forming the required sugar nucleotide. 10 In some embodiments, sugars can be exchanged on a glycosylated natural product or transferred from one glysosylated natural product to another natural product (aglycon exchange) as described in literature.47In some embodiments, sugar nucleotides may be made in a suitable organism, such as 15 in cell, which naturally comprises, or which has been engineered to express, sugar biosynthetic machinery and thymidyl transferase enzyme. As discussed in more detail below, the cell may be a cell that is also capable of producing a polyene. The cell may be naturally capable of producing a polyene, or engineered to express a polyene biosynthetic gene cluster. 20 The polyene substrate for use in embodiments of any of the disclosed aspects may be obtained from commercial sources. The polyene substrate for use in any aspect of the disclosed invention may be obtained 25 from an organism that naturally comprises, or that has been engineered to comprise, a polyene biosynthetic gene cluster (BGC). The BGC may be a previously reported BGC, such as the BGC from S. nodosus or S. noursei. Alternatively, the BGC may be the BGC from S. netropsis or S. kasugaensis as described below and shown in the accompanying figures, and having SEQ ID NO: 14 and SEQ ID NO: 15, respectively. Any suitable strain 30 of organism may be engineered to express a polyene BGC. In some embodiments, the engineered strain may be a strain of organism comprising a polyene BGC, such as, for example, S. netropsis or S. kasugaensis, wherein the strain is engineered to modify the properties of one or more of the enzymes present in the gene 35 cluster. Thus, in some embodiments, the engineered strain may be a strain of organism 126210PCT1 - 74 - comprising a modified polyene BGC. Enzymes that may be modified include polyketide synthase (PKS) and post-PKS modifying enzymes (also called tailoring enzymes). For example, in some embodiments, modifying the polyene BGC may comprise varying 5 the choice of starter unit that selects for acyl-CoA or derivatives in the loading module, thus altering the R2group. An example of this is the replacement of oleandomycin or tylosin loading module with that of erythromycin to give altered products.38Additionally or alternatively, modifying the polyene BGC may comprise the 10 incorporation of alternative malonyl-CoA extender units, which maybe unsubstituted or substituted with methyl, ethyl, methoxy, allyl, benzyl, 5-hexynoyl, haloethyl or substituents which may alter L1, L2or R6group. An example is the synthesis of 2- propargylerythromycin via incorporation of 2-propargylmalonate.3915 In some embodiments, PKS may be engineered to activate or inactivate PKS domains, such as ketoreductase, dehydratase, or enoylreductase, to alter oxidation levels at specific positions on L1and L2. In some embodiments, the method may comprise the expression of hybrid PKS where 20 modules from different PKS are combined together. In some embodiments, the addition or deletion of modules may be used to alter the ring size. An example, of this is the production of an AmB contracted macrolactone.38, 39In some embodiments the polyene substrate for use in in vitro cell-free methods of the 25 twelfth aspect may be extracted from the natural producer strains. Examples of strains that produce polyenes that may be suitable for use in any of the disclosed aspects include Actinobacteria preferably Streptomyces species e.g., S. nodosus (amphotericin B), S. noursei (nystatin), S. albulus (nystatin1056), S. natalensis (pimaricin), S. chattanoogensis (pimaricin), S. diastaticus var 108 (rimocidin, CE108), S. rimosus 30 (rimocidin, CE108), S. lucensis (lucimycin) and S. griseus (candicidin). In addition to Streptomyces, other members of phylum Actinobacteria such Micromonospora, Kitasatospora, Nocardiopsis, Pseudonocardia, Nocardia, Actinoplanes, Saccharopolyspora and Amycolatopsis may also encode polyene 35 biosynthesis machinery, well known examples of which are Pseudonocardia autotrophica species that make nystatin-like Pseudonocardia polyene. 126210PCT1 - 75 - The method may comprise contacting the polyene and the glycosyltransferase in the presence of a nucleotide-linked sugar in vitro (test tube) or in vivo (in a recombinant cell) as described below. 5 The nucleotide-linked sugar may be a compound of formula (X): R7a-L4-R12(X) 10 wherein R7ais a sugar moiety; L4is absent or is a linker; and R12is a nucleotide. R7amay be a monosaccharide, a disaccharide, a polysaccharide or a derivative thereof. 15 R7amay be as defined in relation to the first aspect in relation to R7. O L4may be OH , wherein an asterisk indicates a point of bonding to R12. , wherein R13is a 20 nucleobase. R12may may be thymine, guanosine, uridine or cytosin . The molar ratio of the nucleotide-linked sugar to the polyene may be between 1:2 and 10:1, between 1:1 and 5:1 or between 1.5:1 and 3:1. In some embodiments, the molar 25 ratio of the nucleotide-linked sugar to the polyene may be about 2:1. The polyene and the glycosyltransferase may be contacted at an elevated temperature. The elevated temperature may be between 15 and 40°C, between 20 and 37°C, between 25 and 35°C or between 28 and 32°C. 30 126210PCT1 - 76 - The polyene and the glycosyltransferase or the recombinant cell, and optionally the nucleotide-linked sugar, may be incubated for at least one hour, at least two hours, at least 3 hours, at least 6 hours, at least 12 hours or at least 18 hours. The polyene and the glycosyltransferase or the recombinant cell, and optionally the nucleotide-linked 5 sugar, may be incubated for between 1 hour and a month, between 2 hours and three weeks, between 3 hours and two weeks, between 6 hours and a week, between 12 and 72 hours or between 18 and 24 hours. The present inventors have identified amidotransferase enzymes that are capable of 10 derivatising the carboxyl groups of polyenes. Thus, in accordance with a thirteenth aspect, there is provided a method of producing a polyene comprising a modification at an exocyclic carboxylic acid thereon, the method comprising contacting the polyene with an amidotransferase enzyme in the presence of 15 a nitrogen donor to thereby produce the modified polyene. The method of the thirteenth aspect may produce a polyene of the first aspect. With reference to Formula I, it may be appreciated that the exocyclic carboxylic acid 20 which is modified may be at the R3position. Accordingly, the polyene which is contacted with the amidotransferase in the method of the thirteenth aspect, may be a polyene of formula XI: 25 wherein, R1, R2, R4and L1to L3are as defined in relation to the first aspect. The method of the thirteenth aspect may produce a polyene of formula XI: 126210PCT1 - 77 - wherein R3ais CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor 5 CONR5OOR5a; and R1, R2, R4, R5, R5ato R5band L1to L3are as defined in relation to the first aspect. In some embodiments R3ais CONR5R5a, CONR5NR5aR5bor CONR5OR5a. 10 In some embodiments, the modification is amination. For instance, R3amay be CONR5R5a. Preferably, the polyene produced by the method of the thirteenth aspect is not 15 . The amidotransferase enzyme may be a Class II glutamine amidotransferase (GAT) enzyme. 126210PCT1 - 78 - An amidotransferase enzyme that may be used in the method of the thirteenth aspect to modify an exocyclic carboxylic acid on a polyene may be a polyene carboxamide synthase (“PcsA”) enzyme. The amidotransferase enzyme may be the PcsA enzyme expressed by Streptomyces diastaticus, S. rimosus, S. lavendulae which has the amino 5 acid sequence identified under GenBank accession number WP_003981266.1, and set out in SEQ ID NO: 16: MCGISGWLAFDRDLTKEQATVDAMTGTMAYRGPDAGGTWVDRHVALGHRRLAVIDIEGGTQPMRVDTPNG PVAITYSGEVYNFTELREELRRHGHRFRTASDTEVVLRGYLQWGEALADRLNGMYAFAIWDSRNEKLVMI RDRMGIKPFYFHPTADGVLFGSEPKAILAHPMFKRVIEADGLFELLSVCKTPGHAIWSDMREVRPGSLVV 10 VDRAGLRERTYWKLTTQEHTDDRDTTVTKIRELLEDIVRRQLVADVPQCVLLSGGLDSSSITALSARELA AHGEKVRSFSVDFVGLADNFRPDNMRATPDSPFVHDVADHVGSLHEDIVLPHTALTDPDARRAVLAAKDF PSGLADVDVSLYMLFKAIREHSTVALSGETADELFGGYPWFQDPVAQRAGIFPWMVPVLSEWSKANALGL LNPDLIAMSDLGTYVKDRYSEAVAGVEPLPGEDVQERRMRVMSHLHLTRFLQVLLDRKDRISMAVGLEVR VPYCDHRLVEYVYNTPWAMKSFDGREKSLLRAAAGDLLPRSVVERLKSPYPSTQDPGYAGGLQQMGKQLL 15 SEPDHPIFQLVTRASLDKMVKLDPAKMPDAIREQLDRMMDIATWLEMYQPEIRMS [SEQ ID NO: 16] Thus, the amino acid sequence of the amidotransferase enzyme may consist of, or comprise the sequence identified as SEQ ID NO: 16. 20 The amino acid sequence of the amidotransferase enzyme may comprise substantially the amino acid sequence identified as SEQ ID NO: 16, and / or may consist of or comprise a variant or fragments thereof. The terms “substantially the amino acid sequence”, “variant” and “fragment”, as used herein, unless otherwise specified, refer to 25 a sequence that has a minimum level of sequence identity with the identified sequence. Thus, the amino acid sequence of the amidotransferase enzyme may comprise at least 30% sequence identity, at least 40% sequence identity, at least 50% sequence identity, preferably at least 60% sequence identity, more preferably at least 70% sequence identity, to the sequence identified as SEQ ID NO: 16. 30 Another amidotransferase enzyme that may be used in the method of the thirteenth aspect to modify an exocyclic carboxylic acid on a polyene may be the PcsA homologue from Streptomyces albofacians, which is also referred to as asparagine synthase (glutamine-hydrolyzing), or PcsC. 35 126210PCT1 - 79 - The PcsC enzyme from Streptomyces albofacians has been found to possess at least 75% of the activity of the PcsA enzyme expressed by Streptomyces diastaticus, when used in the method of the thirteenth aspect with L-glutamine as a substrate. 5 Thus, the amidotransferase enzyme may be the PcsC enzyme from Streptomyces albofacians, which has the amino acid sequence identified under GenBank accession number WP_150248585.1, and set out in SEQ ID NO: 17: MCGISGWLAFDHDLTKEQATVDAMTGTMAYRGPDAGGTWVDRHVALGHRRLAVIDIEGGTQPMRVDTPNG PVVITYSGEVYNFTELREELRRHGHRFRTASDTEVVLRGYLEWGEALADRLNGMYAFAVWDSRHEKLVMI 10 RDRMGIKPFYFQPTADGVLFGSEPKAILAHPMFKRVIDADGLFELLSVCKTPGHAIWADMREVRPGSLVV VDRAGVRERTYWKLATQEHTDDRDTTVATIRDLLEDIVRRQLVADVPQCVLLSGGLDSSSITALAARELA AHGDKVRSFSVDFTGLADNFRPDSMRATPDSPYVHDVADHVGSLHQDVVLPYTALTDLDARRAVIAAKDF PSGLADVDVSLYVLFKAIREHSTVALSGESADELFGGYPWFQDPVAQRAGIYPWMVPVMSAWSRASVGGG LNPDLLTTSDLMTYLRDRYSEAVADVEMLPGEDEHERRMRVMSHLHLTRFLQVLLDRKDRMSMAVGLEVR 15 VPYCDHRLVEYVYNTPWSMKSFDGREKSLLRAAAGDLLPQSVVDRLKSPYPSTQDARYAGGLQQMGRQLL SEADHPVFQLVTRSTIDEMVKLDPAKMPDAVREQLDRTIDIATWLDMYQPDIRVS [SEQ ID NO: 17] Thus, the amino acid sequence of the amidotransferase enzyme may consist of, or 20 comprise the sequence identified as SEQ ID NO: 17. The amino acid sequence of the amidotransferase enzyme may comprise substantially the amino acid sequence identified as SEQ ID NO: 17, and / or may consist of or comprise a variant or fragments thereof. Thus, the amino acid sequence of the 25 amidotransferase enzyme may comprise at least 30% sequence identity, at least 40% sequence identity, at least 50% sequence identity, preferably at least 60% sequence identity, more preferably at least 70% sequence identity to the sequence identified as SEQ ID NO: 17. 30 The amidotransferase enzymes disclosed herein that have been found by the inventors to be capable of modifying an exocyclic carboxylic acid on a polyene share the same overall protein structure. The shared protein structure has been found to include two catalytic domains. An N-terminal glutamine hydrolase domain releases ammonia from the nitrogen donor, which travels through a molecular tunnel to the C-terminal ATP- 35 dependent domain that activates the polyene substrate, forming an acyl-AMP intermediate which is subsequently attacked by the ammonia, ultimately modifying an exocyclic carboxylic acid on the polyene substrate. As shown in the following sequence 126210PCT1 - 80 - alignment, the PcsC enzyme from Streptomyces albofacians (SEQ ID NO: 17) has 90% sequence identity to the PcsA enzyme expressed by Streptomyces diastaticus (SEQ ID NO: 16). 5 PcsA 51 LAVIDIEGGTQPMRVDTPNGPVAITYSGEVYNFTELREELRRHGHRFRTA 100 10 ||||||||||||||||||||||.||||||||||||||||||||||||||| PcsC 51 LAVIDIEGGTQPMRVDTPNGPVVITYSGEVYNFTELREELRRHGHRFRTA 100 PcsA 101 SDTEVVLRGYLQWGEALADRLNGMYAFAIWDSRNEKLVMIRDRMGIKPFY 150 |||||||||||:||||||||||||||||:||||:|||||||||||||||| 15 PcsC 101 SDTEVVLRGYLEWGEALADRLNGMYAFAVWDSRHEKLVMIRDRMGIKPFY 150 PcsA 151 FHPTADGVLFGSEPKAILAHPMFKRVIEADGLFELLSVCKTPGHAIWSDM 200 |.|||||||||||||||||||||||||:|||||||||||||||||||:|| PcsC 151 FQPTADGVLFGSEPKAILAHPMFKRVIDADGLFELLSVCKTPGHAIWADM 200 20 PcsA 201 REVRPGSLVVVDRAGLRERTYWKLTTQEHTDDRDTTVTKIRELLEDIVRR 250 |||||||||||||||:||||||||.||||||||||||..||:|||||||| PcsC 201 REVRPGSLVVVDRAGVRERTYWKLATQEHTDDRDTTVATIRDLLEDIVRR 250 25 PcsA 251 QLVADVPQCVLLSGGLDSSSITALSARELAAHGEKVRSFSVDFVGLADNF 300 ||||||||||||||||||||||||:||||||||:|||||||||.|||||| PcsC 251 QLVADVPQCVLLSGGLDSSSITALAARELAAHGDKVRSFSVDFTGLADNF 300 PcsA 301 RPDNMRATPDSPFVHDVADHVGSLHEDIVLPHTALTDPDARRAVLAAKDF 350 30 |||:||||||||:||||||||||||:|:|||:|||||.||||||:||||| PcsC 301 RPDSMRATPDSPYVHDVADHVGSLHQDVVLPYTALTDLDARRAVIAAKDF 350 PcsA 351 PSGLADVDVSLYMLFKAIREHSTVALSGETADELFGGYPWFQDPVAQRAG 400 ||||||||||||:||||||||||||||||:|||||||||||||||||||| 35 PcsC 351 PSGLADVDVSLYVLFKAIREHSTVALSGESADELFGGYPWFQDPVAQRAG 400 PcsA 401 IFPWMVPVLSEWSKANALGLLNPDLIAMSDLGTYVKDRYSEAVAGVEPLP 450 |:||||||:|.||:|:..|.|||||:..|||.||::||||||||.||.|| PcsC 401 IYPWMVPVMSAWSRASVGGGLNPDLLTTSDLMTYLRDRYSEAVADVEMLP 450 40 PcsA 451 GEDVQERRMRVMSHLHLTRFLQVLLDRKDRISMAVGLEVRVPYCDHRLVE 500 |||..|||||||||||||||||||||||||:||||||||||||||||||| PcsC 451 GEDEHERRMRVMSHLHLTRFLQVLLDRKDRMSMAVGLEVRVPYCDHRLVE 500 45 PcsA 501 YVYNTPWAMKSFDGREKSLLRAAAGDLLPRSVVERLKSPYPSTQDPGYAG 550 |||||||:|||||||||||||||||||||:|||:|||||||||||..||| PcsC 501 YVYNTPWSMKSFDGREKSLLRAAAGDLLPQSVVDRLKSPYPSTQDARYAG 550 PcsA 551 GLQQMGKQLLSEPDHPIFQLVTRASLDKMVKLDPAKMPDAIREQLDRMMD 600 50 ||||||:|||||.|||:||||||:::|:||||||||||||:||||||.:| PcsC 551 GLQQMGRQLLSEADHPVFQLVTRSTIDEMVKLDPAKMPDAVREQLDRTID 600 PcsA 601 IATWLEMYQPEIRMS 615 |||||:||||:||:| 55 PcsC 601 IATWLDMYQPDIRVS 615 Since the amidotransferase enzymes expressed by both Streptomyces diastaticus and Streptomyces albofacians have been found to both possess a high level of activity in the disclosed method, the amidotransferase enzyme may consist of or comprise a variant or 126210PCT1 - 81 - fragment of the sequence identified as SEQ ID NO: 16 and / or SEQ ID NO: 17. Thus, the amino acid sequence of the amidotransferase enzyme may consist of or comprise at least 70% sequence identity to the sequence identified as SEQ ID NO: 16 and / or SEQ ID NO: 17. Preferably, the amino acid sequence of the amidotransferase enzyme may 5 consist of or comprise at least 80% or at least 90% sequence identity to the sequence identified as SEQ ID NO: 16 and / or SEQ ID NO: 17. The amino acid sequence of the amidotransferase enzyme may consist of or comprise at least 93%, 95%, 97%, or 99% sequence identity to the sequence identified as SEQ ID NO: 16 and / or SEQ ID NO: 17. 10 The amino acid sequence of the amidotransferase enzyme may comprise a significant proportion, such as at least 80%, 85%, 90%, 93%, 95%, 97%, or 99% of the amino acid residues that are conserved between the sequences identified as SEQ ID NO: 16 and SEQ ID NO: 17. The amino acid sequence of the amidotransferase enzyme may comprise 100% of the amino acid residues that are conserved between the sequences 15 identified as SEQ ID NO: 16 and SEQ ID NO: 17. The amidotransferase enzyme may comprise one or more, such as 2, 3, 4, 5, 6 or 7 amino acid substitutions at positions corresponding to those that are conserved between SEQ ID NO: 16 and SEQ ID NO: 17. 20 The amidotransferase enzyme preferably comprises an Arginine residue in the position corresponding to residue 50 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. 25 The amidotransferase enzyme preferably comprises a Leucine residue in the position corresponding to residue 51 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. The amidotransferase enzyme preferably comprises a Tyrosine or Phenylalanine 30 residue in the position corresponding to residue 76 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. Preferably, the amidotransferase enzyme comprises a Serine or Alanine residue in this position. The amidotransferase enzyme preferably comprises a Serine, Alanine, or Asparagine 35 residue in the position corresponding to residue 77 as defined above in relation to SEQ 126210PCT1 - 82 - ID NO: 16 and SEQ ID NO: 17. Preferably, the amidotransferase enzyme comprises a Serine or Alanine residue in this position. The amidotransferase enzyme preferably comprises a Glycine residue in the position 5 corresponding to residue 78 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. The amidotransferase enzyme preferably comprises a Serine residue in the position corresponding to residue 101 as defined above in relation to SEQ ID NO: 16 and SEQ ID 10 NO: 17. The amidotransferase enzyme preferably comprises an Aspartic acid residue in the position corresponding to residue 102 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. 15 The amidotransferase enzyme preferably comprises a Lysine residue in the position corresponding to residue 147 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. 20 The amidotransferase enzyme preferably comprises a Glutamic acid or Aspartic acid residue in the position corresponding to residue 379 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. Preferably, the amidotransferase enzyme comprises a Glutamic acid residue in this position. 25 The amidotransferase enzyme preferably comprises a Lysine residue in the position corresponding to residue 478 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. The amidotransferase enzyme preferably comprises an Aspartic acid residue in the 30 position corresponding to residue 479 as defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. The amidotransferase enzyme preferably comprises a Glutamic acid residue in the position corresponding to residue 488 as defined above in relation to SEQ ID NO: 16 35 and SEQ ID NO: 17. 126210PCT1 - 83 - The amidotransferase enzyme preferably comprises at least 9 of the following residues at positions in the amino acid sequence corresponding to those defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17: R50, L51, Y76, S77, G78, S101, D102, K147, E379, K479, D479, and E488. More preferably, the amidotransferase enzyme 5 comprises at least 10 or at least 11 of these residues. Most preferably, the amidotransferase enzyme comprises R50, L51, Y76, S77, G78, S101, D102, K147, E379, K479, D479, and E488. Thus, the amino acid sequence of the amidotransferase enzyme may consist of or 10 comprise at least 70% sequence identity to the sequence identified as SEQ ID NO: 16 and / or SEQ ID NO: 17, and also comprise all of the following residues at positions in the amino acid sequence corresponding to those defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17: R50, L51, Y76, S77, G78, S101, D102, K147, E379, K479, D479, and E488. 15 Preferably, the amino acid sequence of the amidotransferase enzyme may consist of or comprise at least 80% or at least 90% sequence identity to the sequence identified as SEQ ID NO: 16 and / or SEQ ID NO: 17, and also comprise all of residues R50, L51, Y76, S77, G78, S101, D102, K147, E379, K479, D479, and E488 at positions in the amino acid 20 sequence corresponding to those defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17. The amidotransferase enzyme may be a mutant or variant of any of the enzymes described herein. For example, the amidotransferase may be engineered to provide 25 increased or otherwise improved activity, or to provide an altered selectivity or specificity for different nitrogen donor and / or polyene substrates. X-ray crystal structures or AlphaFold structural predictions may be generated for the amidotransferase and used to design modifications to the enzyme, for example, by identifying putative nitrogen donor and / or polyene bind sites within the AlphaFold 30 structures. Amino acid residues present in these binding sites may then be subjected to site directed mutagenesis to alter the binding sites and properties of the enzymes. One particularly useful approach for improving the properties of enzymes is the iterative saturation mutagenesis (ISM) method.32 Also, more random mutagenesis approaches such as the well-known error prone PCR method can be used to create libraries of 35 enzymes variants. The mutant enzyme variants may then be screened (tested) for 126210PCT1 - 84 - activity with different polyene and nitrogen donor depending on the preferred modified polyene product that is required. The method may comprise the use of a chimeric amidotransferase. Chimeric 5 amidotransferases may be produced by combining domains, such as a glutamine binding domain from a first amidotransferases enzyme and a synthetase domain from a second amidotransferases enzyme.33, 34 Moreover, chimeric amidotransferases can be engineered by swapping promiscuous 10 glutamine binding domains and synthetase domains from different enzymes. For example, the glutamine binding domain of PcsA-Tag1 could be replaced by a promiscuous domain from a different amidotransferase. The production and use of chimeric amidotransferase enzymes in this way may provide enzymes with altered substrate selectivity that may result in the transfer of an alternative group to the 15 polyene substrate. The amidotransferase enzyme may be a substantially isolated or purified amidotransferase enzyme. The terms “substantially isolated” and “substantially purified” as used herein, unless otherwise specified, are used interchangeably to refer to 20 a product, in this case, a protein, that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it was previously associated. 25 The method may be an in vitro cell-free method. For example, the nitrogen donor and the polyene may be incubated with the amidotransferase enzyme to produce a modified polyene in a biotransformation reaction. Thus, the method may comprise contacting a polyene and a nitrogen donor with a 30 amidotransferase enzyme as defined in accordance with the thirteenth aspect to produce a modified polyene of the first aspect. Indeed, the amidotransferase enzyme that may be used in the method of the thirteenth aspect may be any amidotransferase enzyme that is capable of modifying an exocyclic 35 carboxylic acid on a polyene when contacted with a polyene and a nitrogen donor in an in vitro cell-free method. 126210PCT1 - 85 - The method may comprise the use of a recombinant enzyme. Thus, the method may comprise the expression and purification of a recombinant amidotransferase enzyme, and the subsequent use of the enzyme to produce a modified polyene. Various suitable 5 methods for expressing and purifying an enzyme are known. In some embodiments, the amidotransferase enzyme may be expressed in recombinant form from a suitable cell line or protein expression system. The enzyme may be expressed in E. coli or another host cell using a suitable expression vector, for example, 10 and purified for use in the reaction.36 In some embodiments, the recombinant amidotransferase enzyme may be concentrated and / or purified. Various suitable methods for concentrating and / or purifying an enzyme are known in the art and may be used in the method of the thirteenth 15 aspect.The amidotransferase enzyme may comprise a chemical moiety, referred to herein as an “affinity tag” or “tag”, suitable for use in the affinity purification of the enzyme. The tag may be a polyhistidine tag, consisting of or comprising at least five, such as at least six, at least seven, at least eight, or at least nine histidine residues. The tag may consist of six consecutive histidine residues. 20 Previous attempts to purify amidotransferase enzymes, including those described in relation to the thirteenth aspect failed. To overcome this problem, these enzymes have previously been cooexpressed with a chaperone protein to enable correct protein folding. The present inventors have discovered, however, that previous failures to 25 express amindotransferases comprising an affinity tag failed due to the affinity tag, when present at either the N- or C-termini leads to loss of structure and consequently function, of the enzymes. Having identified this problem, the inventors found that it could be overcome by inserting an affinity label into the middle of the protein sequence, at a position identified using an in-silico homology modelling approach as 30 corresponding to an accessible flexible loop region of the protein. The amidotransferase enzyme may comprise an affinity tag such as a histidine tag in the loop of the folded protein that comprises the amino acid corresponding to residue 70 as defined in SEQ ID NO: 16 and / or SEQ ID NO: 17. For example, the amidotransferase enzyme may comprise an affinity tag such as a histidine tag in the 35 position corresponding to amino acid 67, 68, 69, 70, 71, or 72, as defined in SEQ ID NO: 16 and / or SEQ ID NO: 17. 126210PCT1 - 86 - The finding that an affinity tag may be incorporated into this section of the amidotransferase sequence is somewhat surprising because although this region is hypothesised to comprise an accessible loop structure, there is nevertheless a stringent 5 constraint on this region of the protein sequence, as evidenced by the above sequence alignment showing 100% sequence identity in this region between SEQ ID NOs: 16 and 17. Thus, the amino acid sequence of the amidotransferase enzyme may consist of, or 10 comprise the PcsA sequence, further comprising a histidine tag in the position corresponding to amino acids 68-71. This sequence is referred to as PcsA-Tag1, as shown in SEQ ID NO: 18: MCGISGWLAFDRDLTKEQATVDAMTGTMAYRGPDAGGTWVDRHVALGHRRLAVIDIEGGTQPMRVDTGSS HHHHHHSSGVAITYSGEVYNFTELREELRRHGHRFRTASDTEVVLRGYLQWGEALADRLNGMYAFAIWDS 15 RNEKLVMIRDRMGIKPFYFHPTADGVLFGSEPKAILAHPMFKRVIEADGLFELLSVCKTPGHAIWSDMRE VRPGSLVVVDRAGLRERTYWKLTTQEHTDDRDTTVTKIRELLEDIVRRQLVADVPQCVLLSGGLDSSSIT ALSARELAAHGEKVRSFSVDFVGLADNFRPDNMRATPDSPFVHDVADHVGSLHEDIVLPHTALTDPDARR AVLAAKDFPSGLADVDVSLYMLFKAIREHSTVALSGETADELFGGYPWFQDPVAQRAGIFPWMVPVLSEW SKANALGLLNPDLIAMSDLGTYVKDRYSEAVAGVEPLPGEDVQERRMRVMSHLHLTRFLQVLLDRKDRIS 20 MAVGLEVRVPYCDHRLVEYVYNTPWAMKSFDGREKSLLRAAAGDLLPRSVVERLKSPYPSTQDPGYAGGL QQMGKQLLSEPDHPIFQLVTRASLDKMVKLDPAKMPDAIREQLDRMMDIATWLEMYQPEIRMS [SEQ ID NO: 18] The amino acid sequence of the amidotransferase enzyme may consist of, or comprise 25 the PcsC sequence, further comprising a histidine tag in the position corresponding to amino acids 68-71. This sequence is referred to as PcsC-Tag1, as shown in SEQ ID NO: 19: MCGISGWLAFDHDLTKEQATVDAMTGTMAYRGPDAGGTWVDRHVALGHRRLAVIDIEGGTQPMRVDTGSH HHHHHSGVVITYSGEVYNFTELREELRRHGHRFRTASDTEVVLRGYLEWGEALADRLNGMYAFAVWDSRH 30 EKLVMIRDRMGIKPFYFQPTADGVLFGSEPKAILAHPMFKRVIDADGLFELLSVCKTPGHAIWADMREVR PGSLVVVDRAGVRERTYWKLATQEHTDDRDTTVATIRDLLEDIVRRQLVADVPQCVLLSGGLDSSSITAL AARELAAHGDKVRSFSVDFTGLADNFRPDSMRATPDSPYVHDVADHVGSLHQDVVLPYTALTDLDARRAV IAAKDFPSGLADVDVSLYVLFKAIREHSTVALSGESADELFGGYPWFQDPVAQRAGIYPWMVPVMSAWSR ASVGGGLNPDLLTTSDLMTYLRDRYSEAVADVEMLPGEDEHERRMRVMSHLHLTRFLQVLLDRKDRMSMA 35 VGLEVRVPYCDHRLVEYVYNTPWSMKSFDGREKSLLRAAAGDLLPQSVVDRLKSPYPSTQDARYAGGLQQ MGRQLLSEADHPVFQLVTRSTIDEMVKLDPAKMPDAVREQLDRTIDIATWLDMYQPDIRVS [SEQ ID NO: 19] 126210PCT1 - 87 - Thus, the amidotransferase enzyme may consist of or comprise at least 30% sequence identity, at least 40% sequence identity, at least 50% sequence identity, preferably at least 60% sequence identity, more preferably at least 70% sequence identity, to the 5 sequence identified as SEQ ID NO: 16 and / or SEQ ID NO: 17, and may further comprise an affinity tag in the loop of the folded protein that comprises the amino acid corresponding to residue 70 as defined in SEQ ID NO: 16 and / or SEQ ID NO: 17. Preferably, the amidotransferase may further comprise all of residues R50, L51, Y76, 10 S77, G78, S101, D102, K147, E379, K479, D479, and E488 at positions in the amino acid sequence corresponding to those defined above in relation to SEQ ID NO: 16 and SEQ ID NO: 17, and may further comprise an affinity tag in the loop of the folded protein that comprises the amino acid corresponding to residue 70 as defined in SEQ ID NO: 16 and / or SEQ ID NO: 17. 15 The amidotransferase enzyme may comprise an affinity label and may consist of or comprise at least 30% sequence identity, at least 40% sequence identity, at least 50% sequence identity, preferably at least 60% sequence identity, more preferably at least 70% sequence identity to the sequence identified as SEQ ID NO: 18 and / or SEQ ID NO: 20 19. Preferably, the amidotransferase enzyme may comprise an affinity label and may consist of or comprise at least 80% or at least 90% sequence identity to the sequence identified as SEQ ID NO: 18 and / or SEQ ID NO: 19. In addition, or as an alternative, to the use of a purified enzyme, the method may be an 25 in vitro cell-free method comprising the use of a cell lysate comprising the amidotransferase enzyme. In some embodiments, the method may be an in vitro cell-free method comprising the use of a crude enzyme extract comprising the amidotransferase enzyme. 30 Various approaches are known for preparing recombinant enzymes for use in in vitro cell-free biotransformation reactions, and such methods may be used in the method of the thirteenth aspect. Such methods, include, for example, immobilisation of the amidotransferase enzyme on a suitable solid support, or encapsulation of the 35 amidotransferase enzyme within a porous membrane.37 126210PCT1 - 88 - The nitrogen donor for use in embodiments of the thirteenth aspect comprising an in vitro cell-free method may be obtained from commercial sources. In some embodiments, particularly in embodiments in which the method comprises an 5 in vitro cell-free system, the nitrogen donor may comprise L-glutamine, which may be obtained from commercial sources. In some embodiments, particularly in embodiments in which the method comprises the use of a recombinant organism, the nitrogen donor may be produced intracellularly. 10 For example, in such embodiments, the nitrogen donor may comprise L-glutamine produced intracellularly as an essential amino acid. In some embodiments, the nitrogen donor may comprise a glutamine analogue, which may be synthesised chemically or obtained commercially. 15 The polyene substrate for use in embodiments of the thirteenth aspect may be obtained by any of the approaches described above in relation to the twelfth aspect. The nitrogen donor may be ammonia, an ammonium ion, an ammonium salt or a 20 compound of formula XII: wherein R14is NR5R5a, NR5NR5aR5b, NR5OR5a, NR5NR5aOR5b, NR5OOR5aor 25 NR5CONR5aR5b, wherein R5, R5aand R5bare as defined above. The compound of formula XII may be a compound of formula XIIa: 30 The molar ratio of the amine or compound of formula XII to the polyene may be between 1:2 and 10:1, between 1:1 and 5:1 or between 1.5:1 and 3:1. In some 126210PCT1 - 89 - embodiments, the molar ratio of the amine or compound of formula XII to the polyene may be about 2:1. The polyene and the amidotransferase may be contacted at an elevated temperature. 5 The elevated temperature may be between 15 and 40°C, between 20 and 37°C, between 25 and 35°C or between 28 and 32°C. The polyene and the amidotransferase or the recombinant cell, and optionally the amine or compound of formula XII, may be incubated for at least one hour, at least two 10 hours, at least 3 hours, at least 6 hours or at least 12 hours. The polyene and the amidotransferase or the recombinant cell, and optionally the amine or compound of formula XII, may be incubated for between 1 hour and a month, between 2 hours and a week, between 3 and 72 hours, between 6 and 48 hours or between 12 and 24 hours. 15 The nitrogen donor and polyene can be incubated with the amidotransferase in vitro by a biotransformation reaction. In accordance with a fourteenth aspect, there is provided a method of producing a modified polyene comprising a sugar moiety and comprising a modification at an 20 exocyclic carboxylic acid thereon, the method comprising: - contacting a polyene and a glycosyltransferase enzyme in the presence of a nucleotide-linked sugar; and - contacting the polyene and an amidotransferase enzyme in the presence of a nitrogen donor; 25 to thereby produce the modified polyene. The method may comprise contacting the polyene and the glycosyltransferase enzyme prior to contacting the polyene and the amidotransferase enzyme. Alternatively, the method may comprise contacting the polyene and the glycosyltransferase enzyme after 30 contacting the polyene and the amidotransferase enzyme. Alternatively, the method may comprise contacting the polyene and the glycosyltransferase enzyme simultaneously to contacting the polyene and the amidotransferase enzyme. The method may produce a polyene of the first aspect. 35 126210PCT1 - 90 - Prior to contacting either the glycosyltransferase enzyme or the amidotransferase enzyme, the polyene may be a polyene of formula XIII: 5 wherein, R1, R2, R4, R6, L1and L2are as defined in relation to the first aspect. The method of the fourteenth aspect may produce a polyene of formula XIV: 10 wherein R7ais a sugar moiety; R3ais CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor CONR5OOR5a; and R1, R2, R4to R6, L1and L2are as defined in relation to the first aspect. The sugar moiety 15 may be as defined in relation to the first aspect. Preferably, the polyene produced using the method of the fourteenth aspect is not , 126210PCT1 - 91 - 5 It may be appreciated that the preferred features of the twelfth and thirteenth aspects, recited above, may apply mutatis mutandis to the fourteenth aspect. The method of any of the twelfth, thirteenth, or fourteenth aspects may be a recombinant method, wherein a polyene is produced by a recombinant organism. The 10 recombinant organism may be an organism of one of the strains listed above in relation to the twelfth aspect that has been engineered such that it is capable of producing a polyene as disclosed herein. In some embodiments of the twelfth aspect, a recombinant organism expressing the 15 glycosyltransferase enzyme may be incubated with the nucleotide-linked sugar and the polyene to thereby produce a polyene comprising a sugar moiety. Thus, the method of the twelfth aspect may comprise contacting a polyene and a nucleotide-linked sugar with a recombinant organism expressing a glycosyltransferase 20 enzyme as defined in accordance with the twelfth aspect to produce a polyene of comprising a sugar moiety. 126210PCT1 - 92 - In some embodiments of the thirteenth aspect, a recombinant organism expressing the amidotransferase enzyme may be incubated with the nitrogen donor and the polyene to thereby produce the modified polyene. 5 Thus, the method of the thirteenth aspect may comprise contacting a polyene and optionally a nitrogen donor with a recombinant organism expressing an amidotransferase enzyme as defined in accordance with the thirteenth aspect to produce a modified polyene. 10 In some embodiments of the thirteenth aspect, the nitrogen donor may comprise glutamine produced endogenously within the cell. In such embodiments of the thirteenth aspect, a recombinant organism expressing the amidotransferase enzyme may be incubated with the polyene to thereby produce the modified polyene. Thus, the 15 method of the thirteenth aspect may in such embodiments comprise contacting a polyene with a recombinant organism expressing an amidotransferase enzyme as defined in accordance with the thirteenth aspect to produce the modified polyene. In some embodiments of the fourteenth aspect, a recombinant organism expressing 20 both the glycosyltransferase enzyme of the twelfth aspect and the amidotransferase enzyme of the thirteenth aspect may be incubated with the nucleotide-linked sugar, the polyene, and optionally an exogenous nitrogen donor, to thereby produce the modified polyene. 25 Thus, the method of the fourteenth aspect may comprise contacting a polyene, a nucleotide-linked sugar, and optionally an exogenous nitrogen donor, with a recombinant organism expressing both the glycosyltransferase enzyme of the twelfth aspect and the amidotransferase enzyme of the thirteenth aspect to produce a modified polyene. 30 Recent advances in genome sequencing and bioinformatic tools such as antiSMASH, Prism and MiBig allow the identification of previously unknown polyene encoding clusters. Using this information, the inventors have identified novel polyene pathways and characterised their products such as S. kasugaensis (Kafufungin A, B, C & D) and 35 S. netropsis (mycopentin A, B & C). Similarly, S. albofacians (tetraene) and S. lavendulae encode polyene clusters. Some natural producers encode cryptic clusters 126210PCT1 - 93 - such as Amycolaptosis jejuni which encodes a pentaene cluster that remains silent under laboratory conditions which can be awakened by media screening and modulation of transcriptional control. Any of these strains and / or enzyme clusters may be suitable for producing polyenes for use in the method of the twelfth, thirteenth, 5 and / or fourteenth aspects. In some embodiments of the twelfth aspect, the recombinant organism may be an organism that naturally comprises, or that has been engineered to comprise, a polyene BGC, and further comprises (e.g. that naturally expresses or has been engineered to 10 express) the glycosyltransferase enzyme as defined in accordance with the twelfth aspect. Capturing a natural product pathway and expressing it in a different host is described, for example, in Li, Y., et al. Sci Rep 5, 9383 (2015). In some embodiments, the method may comprise incubating the recombinant organism with the nucleotide- linked sugar to thereby produce the polyene comprising a second sugar moiety. The 15 BGC may consist of, or comprise, the BGC, or a fragment thereof, from Streptomyces nodosus ATCC14899, as described, for example, in Caffrey, P., et al. Cell Chemical Biology 8, 713-23 (2001). In some embodiments, including, in particular, the embodiments discussed below, the BGC may consist of, or comprise, the BGC, or a fragment thereof, from S. netropsis or S. kasugaensis as described below and shown in 20 the accompanying figures and recited in SEQ ID No: 14 and SEQ ID NO: 15, respectively. Thus, the method of the twelfth aspect may comprise contacting a nucleotide-linked sugar with a recombinant organism that naturally comprises, or that has been 25 engineered to comprise, a polyene BGC, and that further comprises (e.g. that naturally expresses or has been engineered to express) the glycosyltransferase enzyme as defined in accordance with the twelfth aspect, to produce the polyene comprising a sugar moiety. 30 In some embodiments of the thirteenth aspect, the recombinant organism may be an organism that naturally comprises, or that has been engineered to comprise, a polyene BGC, and further comprises (e.g. that naturally expresses or has been engineered to express) the amidotransferase enzyme as defined in accordance with the thirteenth aspect. In some embodiments, the method may comprise incubating the recombinant 35 organism, optionally with an exogenous nitrogen donor, to thereby produce a modified polyene. 126210PCT1 - 94 - Thus, the method of the thirteenth aspect may comprise the use of a recombinant organism that naturally comprises, or that has been engineered to comprise, a polyene BGC, and that further comprises (e.g. that naturally expresses or has been engineered 5 to express) the amidotransferase enzyme as defined in accordance with the thirteenth aspect, to produce the modified polyene. In some embodiments of the fourteenth aspect, the recombinant organism may be an organism that naturally comprises, or that has been engineered to comprise, a polyene 10 BGC, and further comprises (e.g. that naturally expresses or has been engineered to express) both the glycosyltransferase enzyme as defined in accordance with the twelfth aspect and the amidotransferase enzyme as defined in accordance with the thirteenth aspect. In some embodiments, the method may comprise incubating the recombinant organism with the nucleotide-linked sugar, and optionally an exogenous nitrogen 15 donor, to thereby produce the modified polyene. Thus, the method of the fourteenth aspect may comprise contacting a nucleotide-linked sugar, and optionally an exogenous nitrogen donor, with a recombinant organism that naturally comprises, or that has been engineered to comprise, a polyene BGC, and that 20 further comprises (e.g. that naturally expresses or has been engineered to express) both the glycosyltransferase enzyme as defined in accordance with the twelfth aspect and the amidotransferase enzyme as defined in accordance with the thirteenth aspect, to produce the modified polyene. 25 In some embodiments of the twelfth aspect, the recombinant organism may be an organism that naturally comprises, or that has been engineered to comprise, sugar biosynthetic machinery, and further comprises (e.g. that naturally expresses or has been engineered to express) the glycosyltransferase enzyme as defined in accordance with the twelfth aspect. In some embodiments, the method may comprise incubating 30 the recombinant organism with the polyene to thereby produce the polyene comprising a sugar moiety. Thus, the method of the twelfth aspect may comprise contacting a polyene with a recombinant organism that naturally comprises, or that has been engineered to 35 comprise, sugar biosynthetic machinery, and that further comprises (e.g. that naturally expresses or has been engineered to express) the glycosyltransferase enzyme as defined 126210PCT1 - 95 - in accordance with the twelfth aspect, to produce the polyene comprising a sugar moiety. In some embodiments of the fourteenth aspect, the recombinant organism may be an 5 organism that naturally comprises, or that has been engineered to comprise, sugar biosynthetic machinery, and further comprises (e.g. that naturally expresses or has been engineered to express) both the glycosyltransferase enzyme as defined in accordance with the twelfth aspect and the amidotransferase enzyme as defined in accordance with the thirteenth aspect. In some embodiments, the method of the 10 fourteenth aspect may comprise incubating the recombinant organism, and optionally an exogenous nitrogen donor, with the polyene to thereby produce the modified polyene. Thus, the method of the fourteenth aspect may comprise contacting a polyene, and 15 optionally an exogenous nitrogen donor, with a recombinant organism that naturally comprises, or that has been engineered to comprise, sugar biosynthetic machinery, and that further comprises (e.g. that naturally expresses or has been engineered to express) both the glycosyltransferase enzyme as defined in accordance with the twelfth aspect and the amidotransferase enzyme as defined in accordance with the thirteenth aspect, 20 to produce the modified polyene. The method of any of the twelfth, thirteenth, and / or fourteenth aspects may comprise the use of an entire polyene BGC, such as a polyene BGC captured from a native polyene producer strain. The method may comprise the transfer of the polyene BGC 25 into a genetically tractable host (Streptomyces, yeast) using Cre-lox recombination system.48or other approaches.49The method may subsequently comprise the delivery of the polyene BGC on integrative or non-integrative plasmids and / or artificial chromosomes to the preferred host strain to thereby produce a recombinant organism. 30 The method of the twelfth and / or fourteenth aspects may comprise the use of an entire polyene BGC, such as a polyene BGC and sugar biosynthetic pathway captured from a native polyene producer strain. The method may comprise the transfer of these genes into a genetically tractable host (Streptomyces, yeast) using Cre-lox recombination system.48or other approaches.49The method may subsequently comprise the delivery 35 of the genes on integrative or non-integrative plasmids and / or artificial chromosomes to the preferred host strain to thereby produce a recombinant organism. 126210PCT1 - 96 - In some embodiments of the twelfth and / or fourteenth aspects, the recombinant organism may comprise (e.g. may have been engineered to express) a polyene BGC, which may comprise a polyketide synthase, a sugar nucleotide subcluster, and the 5 glycosyltransferase enzyme as defined in accordance with the twelfth aspect. In some embodiments of the thirteenth and / or fourteenth aspects, the recombinant organism may comprise (e.g. may have been engineered to express) a polyene BGC, which may comprise a polyketide synthase, and the amidotransferase enzyme as 10 defined in accordance with the thirteenth aspect. In some embodiments, of the fourteenth aspect the recombinant organism may comprise (e.g. may have been engineered to express) a polyene BGC, which may comprise a polyketide synthase, a sugar nucleotide subcluster, a glycosyltransferase 15 enzyme as defined in accordance with the twelfth aspect, and an amidotransferase enzyme as defined in accordance with the thirteenth aspect. The co-expression of the polyene BGC, which may comprise a polyketide synthase, sugar nucleotide subcluster, glycosyltransferase, and optionally other genes, may 20 provide a recombinant organism for use in the method of the twelfth and / or fourteenth aspects, for producing a polyene comprising a sugar. The co-expression of the polyene BGC, which may comprise a polyketide synthase, amidotransferase, and optionally other genes, may provide a recombinant organism for 25 use in the method of the thirteenth and / or fourteenth aspects, for producing the modified polyene. The co-expression of the polyene BGC, which may comprise a polyketide synthase, sugar nucleotide subcluster, glycosyltransferase, amidotransferase, and optionally 30 other genes, may provide a recombinant organism for use in the method of the twelfth thirteenth, and / or fourteenth aspects, for producing the modified polyene. Thus, the method of any of the twelfth, thirteenth, and / or fourteenth aspects may be a fermentation method. Any of the recombinant organisms described herein as being 35 suitable for use in the methods of any of the twelfth, thirteenth, and / or fourteenth aspects may be used in a fermentation method to produce a polyene. 126210PCT1 - 97 - In some embodiments of the twelfth aspect, a glycosyltransferase enzyme as defined in accordance with the twelfth aspect, such as the enzyme from S. netropsis, S. kasugaensis, or S. albulus may be heterologously expressed in a suitable organism, 5 such as S. nodosus (AmB producer), using a suitable expression system, such as pSET152 system that uses a strong ermE* promoter, and the engineered organism used in a fermentation method to produce a polyene. In some embodiments of the thirteenth aspect, an amidotransferase enzyme as defined 10 in accordance with the thirteenth aspect, such as PcsA or asparagine synthase (glutamine-hydrolyzing), may be heterologously expressed in a suitable organism, such as S. nodosus S. noursei, S. albulus, S. natalensis, or S. rimosus, using a suitable expression system, such as pSET152 system that uses a strong ermE* promoter, and the engineered organism used in a fermentation method to produce a modified polyene. 15 In some embodiments of the twelfth, thirteenth, and / or fourteenth aspect, a glycosyltransferase enzyme as defined in accordance with the twelfth aspect, such as the enzyme from S. netropsis, S. kasugaensis, or S. albulus, and an amidotransferase enzyme as defined in accordance with the twelfth aspect, such as PcsA or asparagine 20 synthase (glutamine-hydrolyzing), may be heterologously expressed in a suitable organism, such as S. nodosus S. noursei, S. albulus, S. natalensis, or S. rimosus, using a suitable expression system, such as pSET152 system that uses a strong ermE* promoter, and the engineered organism used in a fermentation method to produce a modified polyene. 25 In any of the embodiments of the method of the twelfth, thirteenth, and / or fourteenth aspects, the BGC may consist of, or comprise, the BGC, or a fragment thereof, from S. netropsis or S. kasugaensis as described below and shown in the accompanying figures. 30 Production levels of glycosylated polyenes in engineered strains maybe optimised by a range of established methods such as re-factoring which involves replacing native promoters with strong constitutive promoter to express desired foreign proteins. Yield optimisation can also be done by metabolic engineering approaches that increase supply of precursors (e.g., sugars, acyl-CoAs, and malonyl-CoA). This can be achieved 35 by introducing genes encoding feed-back resistant variants of key enzymes involved in precursor supply. Alternatively, genes encoding competing pathways (e.g. other 126210PCT1 - 98 - polyketide and sugar pathways) can be deleted using CRISPR Cas9-gene editing or other homologous recombination (gene knockout) approaches. Any suitable method of genetic engineering strains of organism may be used, including, 5 for example, cloning, heterologous expression, gene knock in, gene knock out, gene swapping, all of which have been described and would be familiar to the skilled person. For example, methods for the genetic manipulation of Streptomyces (the prototypical Actinobacterium) can be found in the laboratory manual of Streptomyces.4010 The in vitro (biotransformation) method is convenient for production of modified glycosylated polyenes for initial testing but can involve multiple steps. On the other hand in vivo method can produce glycosylated polyenes and related congeners by a single-step fermentation approach which may be more scalable and thus more suitable of further development and industrial applications. 15 The commercial, natural or engineered (modified) polyene molecules as described above can also be further subjected to chemical modification prior to or after glycosylation (of the twelfth aspect) and / or carboxylic acid functionalisation (of the thrteenth aspect). Several well-known methods for derivatising polyenes such as 20 functionalisation at the internal hemiketal ring, exocyclic carboxylic acid and amino group of the mycosamine moiety exist.41In addition to these, methods for making hydrophosphoryl and fluorinated polyene derivatives have been established.42Examples of synthetic derivatisation of AmB are also described.43, 4425 In accordance with a fifteenth aspect there is provided a recombinant organism as described above, suitable for use in the methods of the twelfth, thirteenth, and / or fourteenth aspect. All features described herein (including any accompanying claims, abstract and 30 drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same 35 may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:- 126210PCT1 - 99 - Figure 1 S. noursei nystatin-associated TDP-deoxysugar subcluster; Figure 2 is a comparison of glucosylation activities of NysSV, MycS3 and KfuSV with AmB and Nys. A) Percentage conversion of 200 µM polyene into the glucosylated derivative. Data is in triplicate with error bars showing standard deviation. B) RP- 5 HPLC chromatogram showing conversion of AmB and Nys into glucosylated derivatives by KfuSV; Figure 3 is an example of the two-step reaction for fucosylation of nystatin. In reaction 1, TDP-L-fucose is formed from L-fucose-1-phosphate and dTTP through the action of the thymidylyltranferase Cps2L. In reaction 2, a polyene substrate (e.g. nystatin, shown 10 here) and the GT (e.g. KfuSV) are added to the crude mixture of reaction 1 to form fucosylated nystatin; Figure 4 A) Sugars tolerated by KfuSV for addition to AmB and Nys, confirmed by mass spectrometry. Proposed structures are drawn with the C35-OH as the predicated position of glycosylation for all. B) Observed accurate masses for AmB sugar derivatives 15 from ESI (positive mode) mass spectrometry. C) Observed accurate masses for Nys sugar derivatives from ESI (positive mode) mass spectrometry; Figure 5 Mycoheptin biosynthetic gene cluster from Streptomyces netropsis; Figure 6 Proposed biosynthesis of the mycoheptin core; Figure 7 LC-HRMS analysis of mycopentin. EIC corresponding to mycopentin m / z 20 924.4951 [M + H]+; Figure 8 LC-HRMS analysis of mycoheptin B and mycopentin B. EICs corresponding to A) mycoheptin B m / z 1082.5530 [M + H – H2O]+ and B) mycopentin B m / z 1084.5687 [M + H]+. EICs are aligned with absorbance trace from diode array detection at 405 nm (mycoheptin) and 350 nm (mycopentin); 25 Figure 9 LC-HRMS analysis of triglycosylated mycopentin. EIC of the proposed triglycosylated mycopentin m / z 1198.6368 [M + H]+, aligned with the absorbance trace from the diode array detection at 350 nm, indicating a pentaene. The proposed structure of this congener is shown, with the two additional glycosyl moieties highlighted in green; 30 Figure 10 A) Kasufungin (Kfu) biosynthetic gene cluster from S. kasugaensis. B) RP- HPLC chromatogram of extracts produced in the 5 different media, showing compounds absorbing at 334 nm. C) Absorbance spectrum of RP-HPLC peaks from S. kasugaensis media screen, showing the triplet absorbance fingerprint associated with pentaenes (318, 334 and 351 nm). D) Proposed structure of kasufungin A, from 35 bioinformatics prediction; 126210PCT1 - 100 - Figure 11 Biosynthesis of kasufungin core macrolactone. Biosynthetically inactive domains are shown in black; Figure 12 A) LC-HRMS analysis of kasufungin A-E. Extracted ion chromatograms (ESI positive mode) showing masses corresponding to [M + H - H2O]+ions. B) Table of 5 observed and calculated masses for kasufungin A-E. C) Proposed structures of kasufungin B, C, D, and E; Figure 13 A) RP-HPLC analysis of a novel glycosylated AmB derivative produced by KasSub expression in S. nodosus (compared to no production in S. nodosus alone or S. nodosus with an empty plasmid). B) Extracted ion chromatogram (ESI positive mode) 10 corresponding to AmB-deoxysugar. C) Accurate mass analysis of AmB-deoxysugar. Calculated m / z = 1054.5581 [M + H]+. D) Extracted ion chromatogram (ESI positive mode) corresponding to AmA-deoxysugar production. E) Accurate mass analysis of AmA-deoxysugar. Calculated m / z = 1056.5738; Figure 14 Alpha fold model of PcsA with docked L-gln and Pim. (A) shows PcsA N- 15 terminal tolerance for L-glu analogues with different side chains and incorporation into the final product in the C-domain. (B) shows L-glu docking into hydrolase domain and important interactions with the surrounding PcsA residues. (C) shows the transit of NH3 (resulting from L-glu hydrolysis) through the channel that connects the N and the C active sites of PcsA. (D) shows further chemical derivitisation of the acyl derivatives 20 obtained using PcsA; Figure 15 Engineering chimeric glycosyltransferases by swapping KfuSV and NysSV1056 sugar and polyene binding domains; Figure 16 MS analysis (ESI +ve) of PcsA catalysed formation of Nys derivatives. Figure shows extracted ion chromatograms of (a) NysNHOH (enzymatic product) with 25 m / z calc.941.5217, obs.941.5218 [M+H]+, (b) NysNH2 (enzymatic product) with m / z calc.925.5268, obs.925.5267 [M+H]+and (c) Nys (substrate) minus enzyme control with m / z calc.926.5108, obs.926.5102 [M+H]+; Figure 17 MS analysis (ESI +ve) of PcsA catalysed formation of Gluco-NysNH2. Figure shows extracted ion chromatograms of (a) Gluco-NysNH2 (enzymatic product) with 30 m / z calc.1087.5796, obs.1087.5796 [M+H]+, (b) Nys-NH2 (substrate) minus enzyme control with m / z calc.925.5268, obs.925.5267 [M+H]+; Figure 18 MS analysis (ESI +ve) of PcsA catalysed formation of Nys1056 derivatives. Figure shows extracted ion chromatograms of (a) Nys1056NHOH (enzymatic product) with m / z calc.1071.5847, obs.1071.5847 [M+H]+, (b) Nys1056NH2 (enzymatic product) 35 with m / z calc.1055.5898, obs.1055.5889 [M+H]+and (c) Nys1056 (substrate) minus enzyme control with m / z calc.1056.5738, obs.1056.5743 [M+H]+; 126210PCT1 - 101 - Figure 191H-NMR of pimaricin carboxamide in CD3OD; Figure 201H-NMR of nystatin carboxamide in CD3OD; Figure 21 SDS gel showing expression of N-terminally truncated PcsA mutants (lysate and soluble fractions); 5 Figure 22 in silico analysis of L-Gln docked PcsA. Figure shows L-Gln ligand (yellow) docked into PcsA glutaminase active site cavity (grey cartoon). Important residues within 5A are highlighted in ball and sticks with different colours. The main catalytic Cys2 is shown in red and other key residues include Arg50, Ser77, Gly78, Tyr76, Asp102, Ser101, and Glu79. Polar interactions are shown by yellow dashed lines. PyRx 10 software was used for the analysis; Figure 23 Residues lining the molecular channels. Figure shows comparison of the residues lining the molecular channels in 1CT9 and PcsA. Negatively charged residues shown in (white), positively charged (magenta), polar (peach) and hydrophobic (green). The residues chosen for mutagenesis are highlighted in red (K147, E379, K478, D479). 15 The channel in PcsA was identified by superimposition of PcsA model and 1CT9 in PyMOL; Figure 24 PcsA mutagenesis (a) shows mutagenesis of the key residues in L-Gln binding N-domain (b) mutagenesis of the channel forming charged residues; Figure 25 shows HPLC traces comparing the stabilities of Nys and Nys1056 under 20 PcsA assay conditions. (ai) shows PcsA catalysed conversion of nystatin to amide derivative which is also unstable over the course of reaction and gives two product peaks (peak1a and peak2a), (aii) shows negative assay control without enzyme and Nys is unstable during reaction as it splits into two peaks (peak1 and peak2), (aiii) shows the commercial Nys (under non-assay conditions). The peaks 2 and 2a are twisted Nys 25 isoforms. (bi) shows PcsA catalyzed Nys1056NH2formation, which is stable over the course of reaction, (bii) shows minus enzyme control (biii) shows the Nys1056 (under non-assay conditions); Figure 26 shows (A) reverse glycosylation and addition of new sugars onto the C19 and C35 positions of AmB; (B) reverse glycosylation of AmB using MycDI and GDPMH 30 to remove C19-Mycosamine; (C) glycosylation of the AmB-aglycone with MycDI and GDP-mannose; and (D) glycosylation of AmB-19-Man with KfuSV and TDP-glucose to produce AmB-19-Man-35-Glu; Figure 27 provides structures of polyene variants; and Figure 28 provides (A) IC50 data with ten different fungal pathogens shows sugar 35 modified AmBS and NysA3 variants with higher activity than NysA1 (highlighted in grey) and the variants with improved activity over AmB (highlighted in bold); (B) IC50 126210PCT1 - 102 - data with ten fungal pathogens shows NysA3 derivatives with better activity than NysA1 (highlighted in grey), also better than AmB (shown in bold); (C) horse blood haemolysis (EC50), used as an indicator of toxicity, comparing NysA3 and its C16 modified derivatives (Nys31 and Nys34) to AmB; (D) the fold improvement in antifungal activity 5 (IC50s) of NysA3 derivatives (blue) compared to AmB (red line); and (E) the fold reduced toxicity of NysA3 and its derivatives (blue) compared to AmB (red). Examples As described below, the inventors first identified new polyene tailoring enzymes, 10 glycosyltransferase (GT), that can selectively add additional sugars to improve solubility and other properties of polyenes. This also led to the discovery of pathways to two new families of natural polyenes, which the inventors named “kasufungins (Kfu)” and “mycopentins (Myc)”. The inventors also developed enzymatic methods that could be used to transform the C16-carboxylic acid moiety of polyenes to alternative 15 functionality. In addition to discovering and characterisation of new tailoring enzymes, pathways and natural polyenes products, new bio-based routes were developed to novel polyene derivatives which exhibit higher solubility, significantly improved antifungal activity and reduced toxicity. 20 Example 1: Discovery of novel GTs from S. noursei (Nys Producer). The occurrence of polyenes with second sugars is relatively rare. In addition to selvamicin (Sel),29two diglycosylated Nys derivatives were reported as very minor products from fermentation of Nys producer, S. noursei30. 25 126210PCT1 - 103 - Early studies, suggest these minor variants (NysA3 & Nys1070) have improved properties compared with the parent Nys. However, the GTs involved in the addition of sugars at C35 of Nys, are not present in the Nys biosynthetic gene cluster and their identity remains unknown. The inventors aimed to identify the GT(s) responsible for 5 C35 glycosylation of Nys, as such enzymes could be valuable tools for derivatization of Nys, AmB and other important polyenes. To identify candidate GTs that could add second sugars to Nys, the inventors mined the genome of S. noursei (ATCC 11455) to identify gene clusters encoding GTs and enzymes 10 required for biosynthesis of deoxysugar donor substrates (e.g. TDP-L-digitoxose or TDP-L-mycarose). Two GT genes were identified, but their presence within erythromycin-like gene cluster, suggests they are not principally involved in polyene glycosylation. A third GT encoding gene was found within a subcluster along with TDP- L-digitoxose biosynthesis genes, which is not associated with any other natural product 15 pathway, suggesting it could be responsible for NysA3. (Fig 1). This subcluster contains all the genes necessary for TDP-L-digitoxose biosynthesis, except a 4-ketoreductase. Several similar subclusters, including one predicted to install L-digitoxose on selvamicin, also lack this reductase activity, suggesting it may be coded elsewhere in the genome. Genes in this subcluster were named based on their homology to the TDP-L- 20 digitoxose biosynthetic genes from other clusters (Fig 1), with the GT named nysSV. The amino acid sequence of NysSV (SEQ ID NO: 1) was then used to perform a BLAST search against the RefSeq database of non-redundant protein sequences. The top hits from this BLAST search were investigated and two additional GTs were selected for testing, mycS3 from Streptomyces netropsis and kfuSV from Streptomyces 25 kasugaensis (Table 1). Table 1: Identification of nysSV homologues from nonredundant ReqSeq database 126210PCT1 - 104 - Example 2: In vitro activity of GTs The three GT homologs, NysSV, KfuSV, and MycS3, were tested in vitro with TDP-D- glucose and AmB and Nys as polyene substrates (Fig 2). All three enzymes were shown to glycosylate both polyene substrates. Notably, KfuSV exhibited the highest activity of 5 the three, glycosylating both AmB and Nys in high yields. MycS3 displayed a higher degree of selectivity, showing high activity with Nys compared to AmB. The fact that NysSV showed lowest activity of the three GTs, with AmB and Nys, does not rule out the involvement of NysSV in the biosynthesis of NysA3. Firstly, very low yields of NysA3 and Nys1070 are produced in S. noursei which may mean C35-glycosylation, 10 arises due to the promiscuous activity of a GT (e.g. NysSV), that may have evolved a different function. Moreover, TDP-D-glucose is significantly different from GT substrates (TDP-L-digitoxose or TDP-L-mycarose) that give rise to NysA3 and Nys1070. 15 As KfuSV was the GT with the highest activity. The TDP-sugar substrate scope of this GT was explored next. This was done in a two-step assay where commercially available sugar-1-phosphates are converted into corresponding TDP-sugars using the thymidylyltransferase Cps2L.46HRMS was used to confirm the production of different TDP-sugars. After conversion to the TDP-sugar, KfuSV enzyme and either AmB or Nys 20 were added to the reaction. The results are shown in Fig.4, where many of the derivatives could be detected by LC-HRMS, but only the glucosylated and fucosylated polyenes were clearly visible via RP-HPLC. These results demonstrate that KfuSV can accept a wide range of TDP-sugars, with different structural characteristics, including larger groups at the 2’ position. 25 Example 3: MycSV comes from the uncharacterized mycoheptin BGC The mycS3 gene is a nysSV homolog with 55% identity, identified in the strain Streptomyces netropsis. S. netropsis has previously been reported to produce the heptaene polyene macrolide mycoheptin,52but no BGC for mycoheptin has been 30 described to date. S. netropsis has also been reported to produce uncharacterised pentaenes.53Mycoheptin is similar in structure to both AmB and nystatin but does not have a second sugar that would account for the presence of the nysSV homolog in this cluster. The mycS3 gene was found to be located in a TDP-deoxysugar gene subcluster, adjacent to a partially sequenced PKS cluster. Due to the low quality of the sequencing 35 data available, the inventors resequenced the genome using PacBio instrument and assembled the genome sequence de novo. This revealed one 115 Kb polyene macrolide 126210PCT1 - 105 - cluster, likely encoding genes to produce mycoheptin (Fig 5). The S. netropsis genome from RIII to SVII of the cluster is provided as SEQ ID NO: 14. The cluster contains 6 PKS genes containing 19 modules, and several tailoring enzymes. 5 The cluster is detailed in the Table below: 126210PCT1 - 106 - Interestingly, although the MycS3 deoxysugar subcluster is housed within the larger BGC, there have been no reports of diglycosylated mycoheptin congeners being produced by S. netropsis. Individual AT, KR, DH and ER domains within the 6 PKS proteins were analysed at the sequence level to predict domain specificity and activity. 5 The module organisation of the myc BGC is very similar to nystatin, as would be expected from the similar structures of the two compounds. The cause of one of the main differences (a keto group at C5, compared to a hydroxyl group at this position in nystatin) is likely due to the inactive KR domain in module 17. The proposed biosynthetic pathway of the core macrolactone is shown in Fig 6. This mycoheptin BGC 10 is the only polyene gene cluster in the S. netropsis genome, despite the fact that this strain has also been described as producing a number of uncharacterised pentaene compounds. The mechanism behind this may be similar to amphotericin biosynthesis- where an inefficient ER domain in module 5 is responsible for the tetraene amphotericin A (where the C28-C29 double bond is reduced) being produced as well as 15 AmB.17Here, if the ER domain in module 4 was partially skipped in the same way, it would cause only partial reduction of the C30-C31 bond, resulting in a mix of pentaene and heptaene products. In the case of the amphotericins, it was hypothesised that a truncated linker region between the DH and ER domain was restricted in movement, resulting in the ER domain being skipped. In an alignment between this linker region 20 in modules 5 of AmphC and NysC (where the ER5 in NysC is fully functional) and module 4 of MycC, it can be seen that MycC is likewise truncated in this linker region. For S. netropsis fermentation extract, the production of both heptaenes and pentaenes was seen in SPG, GYM and FSM. For the main heptaene peak, LC-HRMS analysis was consistent with the known structure of mycoheptin. A mass consistent with the 25 equivalent pentaene version of mycoheptin (i.e. with a single bond at the C30-C31 position) was identified (Fig 7). Several different congeners of both heptaenes and pentaenes were also produced. Due to the presence of the GT and the sugar gene cluster, the inventors suspected that these may be different glycosylated congeners, so the HR-LCMS was investigated for appropriate masses. Both diglycosylated 30 mycoheptin and mycopentin were found, called mycoheptin B and mycopentin B (Fig 8). Mycoheptin B was only found in trace quantities. Another major fermentation product was a pentaene with a mass of 1198.63 Da. This mass does correspond to mycoheptin B with an additional glycosyl moiety (Fig 9), which may originate from a different natural product gene cluster elsewhere in the S. netropsis genome. 35 126210PCT1 - 107 - Example 4: KfuSV comes from a novel polyene macrolide BGC The kfuSV gene is a nysSV homolog with 60% identity, identified in the strain Streptomyces kasugaensis. This strain has been reported to produce several natural products, including the aminoglycoside kasugamycin, but there are no previous reports 5 of this strain producing a polyene macrolide.54The S. kasugaensis genome was investigated at the location surrounding the kfuSV gene, revealing a polyene cluster comprised of 26 genes (Fig 10A). The S. kasugaensis genome from RIII to SVII of the cluster is provided as SEQ ID NO: 15. 10 The cluster is detailed in the Table below: 126210PCT1 - 108 - Many of these genes share significant similarity to those for the nystatin biosynthetic gene cluster in S. noursei. Notably, there is a subcluster encoding the synthesis of a TDP-sugar within the larger BGC, which is very similar to the selvamicin SelSV- 5 containing subcluster and the NysSV-containing subcluster in S. noursei. The fact that this subcluster is present within the main BGC (like selvamicin, and unlike nystatin) suggests that the major product of this cluster is likely a diglycosylated polyene. This BGC encodes for a 15 module PKS (including a loading module), the same number of modules as the selvamicin BGC. These PKS modules are spread across 5 PKS proteins. 10 The cluster also contains two cytochrome P450s, a mycosamine installing GT, and the genes required to synthesise GDP-mycosamine. The resulting polyene produced by this cluster was given the name kasufungin (Kfu), due to the likely antifungal properties of the compound.15Each individual AT, KR, DH, and ER domain of the PKS was analysed at the sequence level to identify domain specificity and activity. All AT domains in this BGC are predicted to be active, as indicated by the presence of an intact active site GHSxG motif. In terms of specificity, AT1, 2 and 9 contain the YASH motif indicative of specificity towards methyl-malonyl CoA, whereas the remaining 11 AT domains contain the 20 malonyl-CoA specific HAFH motif. All but one KR domain (KR11) are likely active, displaying the conserved YxxxN motif (where x is A or G). The KR11 inactivity is likely responsible for the presence of a keto group which leads to the formation of a hemiketal moiety, a conserved feature of most polyene macrolides. Of the 7 DH domains, only DH14 appears to be inactive due to the lack of an invariant tyrosine present in a YGP 25 motif, despite the presence of the active site HxxxGxxxxP motif. Only one ER domain is present in the kasufungin PKS (ER13). This was aligned with the sequence of the active ER domain present in NysJ from nystatin biosynthesis. Due to the similarity of conserved residues known to be important for catalysis, ER13 was presumed active. 126210PCT1 - 109 - Individual modules from kasufungin biosynthesis and their domain composition, activity and specificity were compared to the corresponding modules involved in selvamicin biosynthesis. The majority of the macrolactone core is largely identical between the two, with the main divergence seen in modules 13 and 14. Module 13 in Kfu 5 biosynthesis is predicted to catalyse a full reductive cycle, resulting in a carbon-carbon single bond, whereas selvamicin contains a hydroxyl at this position due to inactive domains (DH13 and ER13). Conversely, module 14 of Kfu is predicted to install a hydroxyl group due to the predicted inactivity of DH14, whereas in selvamicin the equivalent domain is active, resulting in a carbon-carbon single bond. The only AT 10 domain that is not clear is the loading domain, this is predicted to use methylmalonate, however this is also the case in selvamicin, but in that case selvamicin has an ethyl group at that position (indicative of ethylmalonate). Based on the above information, a model of Kfu macrolactone core synthesis was generated, shown in Fig 11. 15 Example 5: Isolation and characterization of kasufungins The S. kasugaensis strain was grown under a variety of fermentation conditions to elicit polyene production. Media was chosen from examples in the literature of polyene producing media with a variety of carbon and nitrogen sources (Table 2). 20 Table 2: Examples of media in which polyene macrolides have been successfully produced. Media composition is detailed 126210PCT1 - 110 - 50 ml cultures using the 5 different media were incubated for 7 days at 30 °C. Extracts from these cultures were analysed using RP-HPLC (Fig.10B). A number of natural products were produced in SFM and SPG media. These peaks displayed the characteristic pentaene absorbance fingerprint of 318, 334, and 351 nm (Fig.10C). 5 Production of pentaenes is consistent with bioinformatic predictions, and it is likely that these different peaks represent congeners of the same compound, as there is only one polyene BGC in the genome. LC-HRMS analysis identified 5 main pentaene compounds based on the most abundant masses. A mass corresponding to the predicted mass of kasufungin (m / z 914.47 [M + H]+) was identified, and named 10 kasufungin A. The four remaining compounds had masses that suggested that they represented kasufungin congeners that had incomplete tailoring actions. The compounds were named kasufungin B, C, D, and E and are described in (Fig 12). Partial structural characterisation of kasufungin B was done using 1D (1H, 13C NMR) and 2D (TOCSY, COSY) and compared to reported NMR for selvamicin.2915 Example 6: Kfu sugar subcluster expressed in Amphotericin producer, S. nodosus Production of glycosylated polyenes in vivo is a very attractive prospect, with direct fermentation and extraction being a scalable method for production. The sugar 20 subcluster from the kasufungin BGC (including genes for the GT and the synthesis of the 2,6-dideoxysugar), KasSub, was cloned into a high expression plasmid pSET- ermE*-KasSub and introduced into S. nodosus with standard conjugation protocols. HR-LCMS analysis of the fermentation extract revealed that these derivatives have observed m / z of 1056.5734 [M+H]+ and 1054.5581 [M+H]+ , corresponding to the 25 mass predicted for AmA-deoxysugar and AmB-deoxysugar, respectively (Fig 13). To confirm their prediction, the inventors isolated the most abundant derivative (AmB- deoxysugar) for 1D and 2D NMR characterisation. The core polyene macrolide structure of AmB-deoxy-sugar was confirmed to be the same as AmB by comparing its 1H NMR spectra with the reported AmB data.55Then, the installation of an additional30 sugar in AmB-deoxysugar was confirmed by the two anomeric signals found in the1H- 13C HSQC corresponding for C1’ (δ 4.47 to δ 96.52) and C1” (δ 4.70 to δ 98.88). The first sugar was found to be a D-mycosamine as reported for AmB. While for the second sugar, ROESY data indicated correlations from H1” (δ 4.71) to 5 signals which corresponds to H2” (δ 1.83 & δ 1.95), H3” (δ 3.78-weak signal), C33 (δ 5.52-weak 35 signal), C35 (δ 3.33), and C40 (δ 1.00), suggesting that this sugar is attached at the C33-C40 region of the polyene macrolide. Among these positions only C35 possesses an 126210PCT1 - 111 - OH moiety that allows glycosylation to occur, hence, the inventors suggest that the second sugar is attached at this position. Based on the1H, and COSY data, the inventors hypothesised that this second sugar is a deoxy sugar, and judging from the coupling constant of J3,4 at 2.96 Hz, this sugar is likely to be a digitoxose. Coupling constant 5 between H4” and H5” at J4,5 = 8.96 Hz further indicated that this sugar is in L- conformation making it an L-digitoxose. Example 7: Identification of Nys1056 from S. albulus fermentation In addition to the well-established Nys producer S. noursei, the inventors also found a 10 NysSV homolog in the strain S. albulus DSM40492. Although this strain was reported to produce Nys, it was not reported to produce any diglycosylated Nys. To explore if S. albulus could produce Nys variants with an additional second sugar, the inventors subjected this strain to fermentation. The methanolic extracts of S. albulus showed three major and a few minor polyene products, detected by characteristic tetraene 15 chromophore (λ 290, 304, 320 nm) using reverse phase HPLC. The major polyene peaks had masses [M+H]+926.5108, [M+H]+1056.5738, and [M+H]+1040.5789, equivalent to the masses of nystatin A1, di-glycosylated nystatin A3 and 10-deoxy nystatin A3, respectively. For each of these, the corresponding [M+H]+- H2O fragments, [M+H]++ Na+and [M+H]++ Na++ OH adducts were also detected. 20 S. albulus has not been reported before to produce diglycosylated nystatins as major fermentation products. Based on the mass, the inventors named Nys A3 like congener from S. albulus as Nys1056. Although observed mass of Nys1056 from S. albulus, is consistent with the addition of digitoxose, as observed in Nys A3, it is also consistent 25 with the addition of olivose as a second sugar; digitoxose and olivose differ only in the stereochemistry of the C3´-hydroxyl. Similarly, the mass of 1040 could correspond to 10-deoxy nystatin A3 or nystatin with amecitose as the C35 sugar; amecitose lacks the C3´-hydroxyl present in digitoxose. 30 NMR characterisation was performed to rule out different possibilities. To the best of the inventors’ knowledge, there is no complete NMR characterisation on the structure of intact Nystatin A3 (NysA3). To date, NysA3 has been characterised by spectroscopic identification of the degraded products. The sugar structures were elucidated by methanolysis of NysA3, where the sugars obtained were methylated before subjected to 35 NMR and GC-MS analysis.56Then in a follow up paper, the position of the second sugar (L-digitoxose) was determined by chemical characterisation.57The inventors obtained a 126210PCT1 - 112 - full LCMS and NMR characterisation of the intact NysA3. The core polyene macrolide structure of NysA3 was first verified by comparing its1H and13C NMR spectra with the reported Nystatin data.302D NMR including COSY, HSQC, HMBC and NOESY experiments included further confirmed the overall structure of NysA3. From these 2D 5 NMR experiments, the inventors found that the 1st sugar on NysA3 is a D-mycosamine attached to C19 as reported in standard nystatin structures. While for the second sugar, the inventors’1 NOESY correlation indicated correlations from C1’’ (δ 4.81) to C34 (δ 2.50), C34-CH3 (δ 1.05) and C35 (δ 3.46) and with C35 being the only position that would allow glycosylation to occur, the inventors confirmed that the second sugar is 10 attached at C35. Based on their1H, and COSY data, the inventors hypothesised that this second sugar is a deoxy sugar, and judging from the coupling constant of J3,4 at 3.2Hz, this sugar is likely to be a digitoxose. Coupling constant between H4” and H5” at J4,5 = 8.88Hz further indicated that this sugar is in L- conformation making it an L- digitoxose. 15 Example 8: Purification and optimisation of amidotransferase PcsA In addition to introducing glycosyl groups, the inventors also aimed to develop enzymatic methods to derivatise the carboxyl group of polyenes which is known to be a key determinant of toxicity.14-16The inventors envisaged that the addition of a second 20 sugar combined with modification to the carboxyl group, could be particularly interesting. To this end, the inventors sought to explore the potential of the amidotransferase PcsA, that installs the C12-amide moiety of rimocidin, a smaller polyene similar in structure to Pim.58,59PcsA is predicted to possess two catalytic domains. An N-terminal glutamine hydrolase domain releases ammonia which travels 25 through a molecular tunnel to the C-terminal ATP-dependent domain that activates the polyene substrate, forming an acyl-AMP intermediate which is attacked by the ammonia (Fig 14). PcsA was shown to transform the C12-CO2H of rimocidin and Pim, to the corresponding amide in vivo and using crude cell free extracts.60However attempts to purify the enzyme failed as tagging either the N- or C-termini leads to loss 30 of function. To address this, the inventors used an in-silico homology modelling approach, to guide insertion of an His-tag into more flexible loop regions. Several loop regions were targeted for His-tag insertion, PcsA-Tag1 (T67-V72), PcsA-Tag2 (D234-T235), PcsA- 35 Tag3 (A297-N304), PcsA-Tag4 (F300-F301) and PcsA-Tag5 (D234-T235). Following co-expression with chaperons and purification by Ni-NTA chromatography, the eluted 126210PCT1 - 113 - samples of PcsA-Tag1-5 were analyzed by SDS-PAGE. The 69 kDa PcsA variants were expressed and eluted, suggesting sufficient accessibility of His-tags within loop regions. The chaperons (GrpE-22 kDa, DnaJ-41.1 kDa and DnaK-70 kDa) were also co-eluted. The elutions (PcsA-Tag1-5) were tested for activity against Pim. Significant activity was 5 displayed by the Tag1 variant with internal tag in the N-domain surface loop, whereas no activity was observed for Tags 2, 3, 4 and 5. Assays were optimized to give up to 95% conversion using 50 μl reaction with 12.5 μl (50 % v / v) concentrated PcsA-Tag1 solution, 100 μM pimaricin, 2.5 mM L-gln, 25 mM NaCl, 10 mM MgCl2, 4 mM ATP and 125 mM Tris, pH 7. 10 Example 9: N-terminal Substrate Scope of PcsA-Tag1 As a member of GAT family PcsA performs L-glutamine hydrolysis in the N-domain releasing ammonia which passes through the tunnel and modifies activated polyene- AMP intermediate. The inventors tested L- and D- forms of asparagine and D-Gln as 15 other potential substrates and found that PcsA is extremely stringent towards L-Gln, preferring it as a nitrogen donor. The fact that decreasing the side chain length by only one carbon by using L-asparagine results in near abolished activity is striking and suggests that active site residues are critically positioned to bind the glutaminyl side chain specifically. For this, the inventors tested PcsA-Tag1 with seven L-Gln derivatives 20 (L-glutamic acid γ-monohydroxamate, L-glutamic acid γ-hydrazide, glutamyl methylamide, L-theanine, L-glutamine methylamide, L-glutamine methyl ester hydrochloride and L-glutamine ethyl ester hydrochloride). All reactions were carried out with Pim. RP-HPLC and HRMS / MS analysis confirmed the formation of Pim- NHOH [calc. m / z [M+H]+681.3229, obs.681.3211] and Pim-NHNH2 [calc. m / z 25 [M+H]+680.3389, obs.680.3370] with low activity observed for the latter. The rest of the L-Gln analogues did not show activity with PcsA. In addition to improving the properties of Pim and other polyenes, the resulting acyl derivatives (e.g. Pim-NHOH & Pim-NHNH2) could be subjected to further chemical 30 derivatisation. For example, with reference to Fig 14D, hydrazides 1 are less basic, but more nucleophilic than amines (alpha effect) and are widely used in biorthogonal conjugation reactions42reacting rapidly with aldehydes or ketones, to form stable hydrazones 2 which can also be reduced under mild aqueous conditions (3). Hydrazides also form acyl azides 4 in situ (NaNO2, buffer) that can be converted to 35 functionalised amides 5;62using an excess of a primary amine to avoid reactions with the amino group of mycosamine. Under non-aqueous conditions the acyl azide can 126210PCT1 - 114 - undergo a Curtius rearrangement to the isocyanate 6 forming ureas 7 with amines.63Previously this approach was used to transform the C16-carboxylate of AmB to urea derivatives with improved properties.16However, this required 3-4 chemical steps, protection / deprotection and a toxic reagent (DPPA) is used to generate the acyl azide. 5 Starting from hydrazides, it should be possible to generate ureas in a single step, avoiding toxic reagents and protecting groups. Hydroxamic acids can also undergo derivatisation, Lossen rearrangements or metalation. Example 10: C-terminal Polyene Substrate Scope of PcsA-Tag1 10 PcsA shows a flexible polyene scope by accepting Rim, CE-108 and Pim.64Given the fact that PcsA can accept L-Gln, L-glutamic acid γ-monohydroxamate and L-glutamic acid γ-hydrazide , the inventors aimed at testing its polyene tolerance (Table 3). Table 3: PcsA N- and C-terminal substrate scope 15 The inventors’ findings indicate that in addition to Pim, PcsA also converts 4,5- desepoxyPim-NHOH, rimocidin and related compound CE108 to their hydroxamic acid products. HRMS analysis revealed the masses for Rim-NHOH (calc. m / z [M+H]+783.4274, obs.783.4287) and CE108 (calc. m / z [M+H]+755.3961, obs.755.3975). 20 126210PCT1 - 115 - PcsA has been previously reported to only accept smaller polyenes. To the inventors’ surprise, nystatin showed good conversion (~35-40%) to its amide (obs. m / z [M+H]+925.5267) and hydroxamic acid (obs. m / z [M+H]+941.5218) products (Fig 16). 5 This promising result encouraged the inventors to test a di-glycosylated gluco-nystatin substrate. This is a non-natural nystatin derivative with a second sugar (glucose) installed via a glycosyltransferase, nysSV which was amidated upon reaction with PcsA to give Nys-glc-NH2 (obs. m / z [M+H]+1087.5767) (Fig 17). In addition to this, Nys1056 was converted to its amide and hydroxamic acid derivatives with observed 10 masses; Nys1056-NH2 (m / z [M+H]+1055.5889) and Nys1056-NHOH (m / z [M+H]+1071.5847), respectively (Fig 18). In addition, disaccharide containing Pseudonocardia polyene (NPP) was converted to hydroxamic acid product in low yields. The findings suggest PcsA has a promiscuous nature accepting large polyenes like Nys 15 as well as their di-glycosylated forms. Example 11: Structural Characterization of Amide Polyene Derivatives Based on NMR analysis of RimNH2 it appears that PcsA acts on Pim at C12.65However, there is no confirmed data available on the structure of a PcsA derivatised pimaricin 20 carboxamide analogue. Its homologue, PcsB, (87.2% identity) has been reported to act on the exocyclic carboxyl of pimaricin to produce AB-400, which has been structurally confirmed by1H,13C, and DEPT experiments on N-acetyl-AB-400.66Owing to the low natural abundance (0.36%) of NMR active nitrogen,15N-labelled Pim15NH2 was synthesised and confirmed by combination of 2D1H-1H COSY,1H-15N-HSQC and1H- 2515N-HMBC NMR experiments, conducted in CD3OD, 10% D2O at 800 MHz. The chemical shifts for Pim15NH2 are shown in Fig 19. Using the HSQC and HMBC NMR experiments,15N signal was detected at 114 ppm and the two amide protons at 7.0 and 7.8 ppm, showing correlation with proton at C12in Pim15NH2. 30 In another experiment, Pim, PimNH2 and PimNHOH were compared by observing differences in the chemical shifts of the C27 carbonyl carbon. For this, two experiments were performed, (1) CH-HSQC to identify C12 positions, (2) CH-HMBC to link C12 protons to C27 carbonyl carbon. Chemical shifts for hydroxamic acid carbonyl were recorded at 170 ppm, amide carbonyl at 176 ppm and carboxylic acid carbonyl at 180 35 ppm. These values are within the expected range ~ 150-185 ppm for carboxylic acid and its derivatives. 126210PCT1 - 116 - In contrast to the previous studies, the inventors have shown that PcsA can accept large polyenes such as Nys. For detection purpose,15N-labelled Nys15NH2 was synthesised and confirmed by NMR experiments including1H,15N1H-HSQC, CH-HSQC, CH- 5 HMBC, HNCO and NOE in CD3OD at 800 MHz frequency. The1H NMR and the chemical shifts for Nys15NH2 are shown (Fig 20). The1H-HSQC analysis shows15N signal at 113.2 ppm and the amide1H signals at 6.95 and 7.7 ppm. HNCO links1H at 6.95 ppm with carbonyl frequency at 176 ppm while NOESY links the second amide1H at 7.7 ppm to position H16 on nystatin which was confirmed by CH-HSQC. 10 Example 12: Glutamine Independent Activity and N-terminal Truncations Most of the class II GATs possess both L-glutamine and ammonia dependent activity, free ammonium ions were tested at high concentrations by including ammonium acetate in assays with Pim. Moderate activity (~40% conversion) was observed using 15 free NH4+which increased slightly when the ammonium acetateconcentration was doubled. A control experiment without PcsA-Tag1 confirmed that the incorporation of free NH4+was enzymatic. However, L-Gln dependent activity (95% conversion) is preferred. At this stage, it is unclear whether NH4+diffuses directly into the C-terminal active site or via the N-terminal active site and through the molecular tunnel. 20 Knowledge of this could guide the use of only the C-terminal domain through N- terminal truncation and free NH4+to generate amido polyenes. For this, thirteen N- terminal truncation mutants were tested, each named after the starting amino acid (Table 4). 25 Table 4: PcsA N-terminal truncations 126210PCT1 - 117 - SDS PAGE analysis revealed N69, A115, H93, G114, N122, T225, G112, G78 mutants were mostly insoluble whereas S162, D11 and K174 contained relatively higher proportion of soluble protein (Fig 21). Assays were performed with ammonium acetate 5 using lysate and the soluble fractions, but no activity was observed. N-terminal truncations therefore result in insoluble inclusion bodies or inactive proteins suggesting the important role of N-terminus residues in maintaining structural integrity and activity of PcsA. 10 Example 13: PcsA-Tag1 Engineering – Mutagenesis The promiscuous nature of PcsA makes it an attractive tool for polyene tailoring. However, the limited activity with glutamyl hydrazide and inability to accept other L- Gln analogues demands for expansion of the N-terminus substrate scope. Given the structure of PcsA remains unresolved, bioinformatics tools were used to guide 15 engineering. BLAST search of PcsA against reported crystal structures in the PDB show AsnB from E. coli (1CT9) as the top hit sharing 31% structural identity. Structural alignment of the two proteins further supported by docking studies was used to obtain L-Gln bound PcsA. Important residues within 5 angstroms of the N- domain bound ligand weremutated (Fig 22). The PcsA variants were expressed as soluble proteins, 20 tested individually with L-Gln and its analogues as discussed in section 2, using Pim. The conserved L51 and G78 residues, known to make ligand backbone interactions, were substituted with Pro and Ala. Pro was an interesting substituent as it can often cause kinks in the alpha helices,67resulting in active site bulge that could be beneficial or disruptive to enzyme function and may also alter solvent accessability around the 25 kink.68The four mutants L51A, L51P, G78A, G78P lost activity. Tyr76 side OH is suggested to interact with L-Gln side chain. This was changed to Y76A, Y76F, Y76Q, and Y76V. Y76F retained 90% of the wild type activity while activity was lost with the Y76A, Y76Q and Y76V mutants. Tyr76 appears to have a structural role in maintaining active site shape and is preferred over Phe. S77 in the vicinity of bound ligand was 30 mutated to S77A, S77D and S77N (N being conserved in related family members). Surprisingly, S77A retained the same activity as that of the wild type (active only with L-Gln, L-glutamic acid γ-monohydroxamate and L-glutamic acid γ-hydrazide). The activity was reduced with S77N (~25% conversion with L-Gln) while S77D abolished activity suggesting a preference for smaller residues at this position. The S101 and D102 126210PCT1 - 118 - residues make polar contacts with the ligand, mutations to S101A and D102A were found inactive. The bulkier L-Gln side chain groups compared to NH3 may experience steric hinderance in the channel. Enzyme molecular channels are often lined with non- polar uncharged amino acids to facilitate the transit of leaving groups (NH3 in case of 5 Asn synthetases).69In contrast to 1CT9, PcsA channel is composed of charged residues (K147, K478, D479, E379), the roles of which were investigated by mutating to K147A, K147I, K478A, K478I (I is equivalent of K147&K478 in 1CT9), D479A, D479N (N conserved in 1CT9) (Fig 23). Activity was abolished for all these mutants indicating these residues are important for activity. It would not be surprising to think that the 10 flexible side chain of K147 is serving as a dynamic cap to the channel which lets NH3 in after release from the glutaminase active site directing it down into the synthetase domain through charge repulsion. The occurrence of such gated channels is common in bifunctional enzymes where the product formed in one active site serves as a substrate for the second active site. The channel caps prevent premature intermediate leakage 15 into the environment. An example of such an enzyme is glucosamine-6-phosphate synthase where Trp regulates intermediate movement between the two domains.70E379 is the only negatively charged residue located at the base of the channel that remains conserved in other asparagine synthetase members.13The inventors hypothesized E379 could be involved in salt bridge formation with the incoming 20 ammonium ion where the presence of a negative charge could be crucial for such an interaction. This was mutated to E379A, E379Q, E379N and E379D. The E379N will show the importance of negative charge. E379D being smaller while retaining negative charge may create space for ligands with bigger side chain groups. The E379D mutant retained around 50% activity, while an almost complete loss of activity was observed for 25 the E379N and E379Q variants. The retention of some activity with E379D while complete loss with the rest highlights the importance of the negative charge. This knowledge of important residues will guide further engineering leading to PcsA variants with bettter activity. The results of PcsA mutagenesis are summarized in Fig 24. 30 Example 14: Reverse glycosylation and addition of alternative sugars onto polyene aglycones Changing the sugar moieties of polyenes The inventors’ research led to a method for changing the structure of the existing sugar 35 moiety on the polyenes (R4) in combination with the addition of a second sugar (R7) and / or modifications to the carboxyl group typically present in polyenes (R3) within the 126210PCT1 - 119 - general formula X or Y (below). Changing R4, R7and / or R3substituents of polyenes (formula X or Y) in a combinatorial fashion results in polyene variants with improved properties. 5 R3, R4and R7correspond with the C16, C19 and C35 positions in polyenes that share the same ring size as AmB but are numbered differently in polyenes with smaller ring sizes. For example, the existing sugar (R4) of amphotericin B (AmB) is aD-mycosamine sugar at position C19. The examples below show how the structure of the C19 sugar of AmB can be altered in combination with the addition of a second sugar at C35 (R7) and / or 10 modification of the C16 (R3) carboxylate. The R4 sugar is important for the activity of all polyene molecules. This is evident from 15 the fact that previously, removing the C19-mycosamine of AmB resulted in complete loss of antifungal activity (51). In addition to antifungal activity, the R4sugar is essential for polyene solubility. Enzymatic reverse glycosylation to produce polyene aglycones 20 As described above, genome mining led to the discovery of novel polyene pathways from Streptomyces kasugaensis and Streptomyces netropsis producing kasufungins and mycopentins (or mycoheptins), respectively. Two glycosyltransferases, KfuDI and MycDI, were discovered in S. kasugaensis and S. netropsis, which are very useful for removing and adding sugars to the R4position of polyenes. Both enzymes show around 25 75% sequence identity to AmphDI, known to add mycosamine to the C19 position of amphotericin B (AmB). Based on this homology, we proposed that KfuDI and MycDI are responsible for the addition of mycosamine to the equivalent positions (R4) of the novel polyenes we identified, namely kasufungins and mycopentins / mycoheptins. The glycosyltransferases (GTs) are reversible enzymes, i.e. while these enzymes can add a 30 sugar to polyenes, they are also capable of removing a sugar from polyenes in the presence of excess nucleotide diphosphate (NDP) (47). However, the reverse reaction is not very efficient, as the equilibrium of the reaction favours the forward direction 126210PCT1 - 120 - (addition of sugar), resulting in a very low yield (ca.10%) for removal of the sugar and formation of the polyene aglycone. The inventors have developed a more powerful approach to enzymatically remove 5 sugars from polyene molecules at R4and then selectively install new R4and R7sugar substituents or modifications at R3 (Fig.26A). The inventors’ method uses GTs, initially operating in the reverse direction, with a second enzyme, GDP-mannose hydrolase (GDPMH), or a related NDP-sugar hydrolase. GTs work in the forward direction by transferring the sugar from an NDP-sugar to an alcohol group of the 10 substrate. For example, AmphDI uses GDP-mycosamine (GDP-Myc, Fig.26A) to add the mycosamine sugar to the C19-hydroxyl of the AmB precursor (aglycone). If AmB and excess GDP are incubated with AmphDI or a related GT, the C19-mycosamine can be transferred back to GDP in a reverse glycosylation reaction, but only in very low yields. For example, reverse glycosylation of AmB with AmphDI gives a 5% yield of the 15 AmB-aglycone. The inventors show for the first time that addition of GDPMH can significantly increase the yield of reverse glycosylation reactions, driving the reaction in the opposite direction to that favoured in nature. Moreover, the new GTs, particularly MycDI, are 20 more stable and exhibit much higher catalytic activity than the AmphDI and other related GTs. Incubating MycDI or KfuDI with AmB and GDP in the presence of GDPMH gives the AmB aglycone in a very high yield (80-90%) without the need for any chemical steps (Fig.26B). The GDPMH enzyme functions by hydrolysing the GDP- sugar that is formed in the reverse glycosylation, and in doing so, it prevents the 25 reattachment of a sugar to the polyene molecules (driving the equilibrium in favour of the aglycone). Moreover, hydrolysis of the GDP-Myc by GDPMH generates GDP, which can be recycled, enabling further reverse glycosylation. Consequently, sub- stoichiometric (catalytic) quantities of GDP can be employed. Given that GDP is expensive, this can significantly reduce the costs of such a process for large-scale 30 implementation. Glycosylation of polyene aglycones The aglycone formed as described above can then be incubated with GTs including MycDI or KfuDI in the presence of the desired GDP-sugar to catalyse the forward 35 glycosylation reaction, adding an alternative sugar to C19. For example, incubating AmB-aglycone and GDP-mannose with MycDI results in the addition of mannose at 126210PCT1 - 121 - C19, forming the new AmB-19-Man product in 95% yield (Fig.26C). The intermediate AmB-19-Man can then be incubated with a TDP-sugar in the presence of KfuSV (or other C35-selective GTs NysSV, NysSV1056, MycS3 described earlier) to catalyse the glycosylation of the C35 position. The inventors found that KfuSV is most efficient at 5 adding C35-sugars to polyenes such as AmB-19-Man. For instance, incubating AmB-19- Man with TDP-glucose and KfuSV gives a polyene with two new sugars, AmB-19-Man- 35-Glu, in 90% yield (Fig.26D). The enzymatic cascade reaction from AmB through to AmB-19-Man-35-Glu can be carried out using crude GTs (cell-free lysate), without purifying any intermediates, which is ideal for scale-up. 10 There are many existing methods that can be used to produce GDP- or TDP-sugar molecules, using chemical synthesis, enzymatically, or by a combination of enzymes and chemical synthesis. Given the easy availability of many different GDP- and TDP- sugars, the method described above can be used to produce a wide range of new 15 polyene derivatives with multiple differed glycosyl substituents. Moreover, by combing the methods described in this example with methods described earlier (e.g. examples 2, 9 & 10), it is possible to produce large numbers of polyene derivatives modified at R3, R4, and R7 (formula X and Y), which include variants with improved activity, reduced toxicity, and higher aqueous solubility. 20 Example 15: Antifungal Activity and Cytotoxicity of Polyene Derivatives. The antifungal effects of GT / PcsA derivatised polyenes were tested on Aspergillus fumigatus A116, Aspergillus fumigatus TR34, Candida albicans and Candida auris and represented by MIC values. Additionally, the ability of polyenes (Nys, NysNH2, 25 Pim, PimNH2and Pim-NHOH) to lyse defibrinated horse blood erythrocytes was also analysed as an indicator of toxicity (Table 5). Table 5: Antifungal activity of polyene derivatives 126210PCT1 - 122 - MIC is the lowest drug concentration sufficient to prevent fungal growth, lower values represent the drug is more active. Whereas HC refers to the ability of drug to lyse red blood cells. Higher hemolytic values translate into higher toxicity. AmB-deoxysugar is a 5 promising candidate with antifungal activity comparable to AmB. The addition of glucose to both AmB and Nys reduced the antifungal activity, although this addition may improve other properties (toxicity, solubility). Kasufungin B had a similar but slightly improved antifungal activity compared to selvamicin, consistent with the fact that kasufungin B has an exocyclic carboxylate and a mycosamine moiety, both shown 10 to be important for antifungal activity. PimNH2and Pim-NHOH show 2-fold higher activity against C. auris compared to Pim with 1.5-fold reduced toxicity. With reference to the Nys series, an even more pronounced improvement in the antifungal activity is observed with amide products. Nys-NH2(Nys11) shows up to 4- 15 fold improvement in activity against strains of Aspergillus and Candida with 2.7-fold 126210PCT1 - 123 - reduced toxicity compared to Nys (NysA1). Its amide derivative (Nys1056-NH2, Nys31) is 8-fold more bioactive against (A. fumigatus strains) and 4-fold against (C. albicans, C. auris) compared to Nys1056 (NysA3) with an up to 16-fold improvement compared to Nys (NysA1). In terms of toxicity, Nys-NH2 (Nys11) and 1056Nys-NH2 (Nys31) 5 showed 2.7 and 1.5-fold reduced hemolysis compared to the non-amidated parent compounds, respectively. Considering these observations, polyene amide and hydroxamic acid derivatives show superior properties with Nys1056NH2(Nys31) being the most promising candidate. 10 Example 16: Additional Antifungal Activity and Cytotoxicity of Polyene Derivatives Expanded and improved bioactivity data is shown in Figures 27 and 28. IC50s were determined for AmB derivatives with an additional C35-L-digitoxose sugar (AmBS) and 15 NysA3 with ten clinically relevant fungal pathogens defined as critical or high risk by the WHO, including multi-drug-resistant species (Fig 27), and are provided in Table 6. Table 6: Antifungal activity IC50 (μg / mL) data of polyene derivatives with ten different fungal pathogens. Horse blood haemolysis (EC50, μg / mL) data is also shown with 20 higher values indicating lower toxicity 126210PCT1 - 124 - Table 6 (continued): Antifungal activity IC50 (μg / mL) data of polyene derivatives with ten different fungal pathogens. Horse blood haemolysis (EC50, μg / mL) data is also shown with higher values indicating lower toxicity 5 The IC50s indicate that the addition of L-digitoxose increases antifungal activity against eight of the ten pathogens tested for AmB, including Candida albicans (2.7 fold) and Fusarium oxysporum (2 fold) (Fig 28A). NysA3 is also more potent than the parent NysA1 with six of the pathogens tested, most significantly with Fusarium solani (10.2 10 fold) (Fig 28A). The new polyene variants with an additional C35-sugar combined with 126210PCT1 - 125 - C16-carboxylate modifications are shown to exhibit both improved activity and reduced toxicity (Fig 27, Fig 28B and Fig 28C). The inventors observe that nystatin variants combining C35-sugar and C16- 5 modifications (Nys31, Nys32, Nys33 and Nys34) exhibit higher antifungal activity against most fungal pathogens tested than either parent molecule NysA1 or NysA3, and in several cases are more potent than AmB (the most effective clinically relevant polyene antifungal agent). Notably, Nys33 is 2.5- and 5-fold more potent than clinically relevant AmB and NysA1, respectively, when tested against drug-resistant Aspergillus 10 fumigatus (TR34) (Fig 28D). The NysA3 derivatives also show 2- to 4-fold better activity than NysA1 with the Fusarium species tested (Fig 28D). Moreover, all the dual functionalised nystatin variants (Nys31, Nys32, Nys33 and Nys34) exhibit reduced haemolysis compared with both NysA3 and AmB, indicating these derivatives also have lower toxicity to mammalian cells (Fig 28E). 15 Example 17: Solubility and Stability of Nys1056 over Nys Glycosylation of natural products is known for improving dissolution. For instance, nystatin analogue (NPP) with an additional N-acetyl-glucosamine at C19 as well as arabinogalactan conjugated AmB have improved solubilities.71,72Under tested 20 conditions Nys1056 and Nys showed 217 μg / mL and 93 μg / mL aqueous solubility. The addition of second sugar improves solubility of Nys1056 by 2.3-fold (Table 7). Table 7: Comparison of the solubilities of Nys & Nys1056 25 Nys1056 variant is more stable in aqueous solution than parent Nys, which is known to undergo conformational isomerization around C28-C29 single bond. Consequently, Nys can interconvert between a linear (anti) conformation which is bioactive and a twisted (gauche) conformation which is 4 times less active (ca.70:30 at equilibrium) (Fig 25a).22In contrast Nys1056, does not exhibit any isomerisation and exists 30 exclusively in the bioactive conformation (Fig 25b). 126210PCT1 - 126 - Summary Polyenes are natural products, used to treat many infectious diseases. Here is described the discovery of two new families of polyene antifungal agents, which the inventors 5 have named the kasufungins (Kfu) and mycopentins (Myc). Novel glycosyltransferase (GT) enzymes from Kfu, Myc and other pathways were also characterised. The new GTs were shown to glycosylate various non-native polyene substrates including WHO essential medicines amphotericin B (AmB) and nystatin 10 (Nys). The inventors also succeeded in overproducing, purifying and expanding the substrate scope of an amidotransferase PcsA. In addition to accepting smaller polyene substrates, the inventors have showed PcsA can also accept larger substrates, including Nys 15 generating C16-amides (Y = CONH2). Moreover, the inventors showed PcsA can accept glutamine derivatives with modified side chains (-CONHOH or -CONHNH2), transferring hydroxylamine or hydrazine to generate novel polyene derivatives with hydroxamic acid and acyl hydrazine functionality. 20 Finally, the inventors showed that introducing amide functionality improves significantly antifungal activity and reduces toxicity of polyenes, whilst addition of a sugar improves solubility. Moreover, combining GT and PcsA to add both sugar and amide groups has a very dramatic effect, leading to a polyene derivative with improved solubility possessing up to 16-fold higher antifungal activity than WHO essential 25 medicine AmB or Nys and lower toxicity. Methods In vitro GT activity assays. For assays with TDP-glucose, 5-20 μM GT enzyme, 2 mM TDP-glucose, 1 mM MgCl2, 100 μM polyene substrate, 50 mM Tris HCl, pH 7.4, 30 were mixed in a total reaction volume of 50 μl, and incubated at 30 °C for 24 hrs. For TDP-sugars other than TDP-glucose, an initial step was added where the TDP-sugar is produced from the corresponding sugar-1-phosphate. In this first step, 2 mM dTTP, 2 mM sugar-1-phosphate, 2.2 mM MgCl2, 50 μM Cps2L and 50 mM Tris HCl, pH 7.4 were combined in 50 μl volumes. Assays were incubated for 24 hrs at 37 °C in a shaking 35 thermomixer. After 24 hrs the temperature was adjusted to 30 °C and 5-20 μM of the GT and 100 μl of polyene were added. Assays were then incubated for a further 24 hrs 126210PCT1 - 127 - at 30 °C. For all assays (1-step or 2-step), reactions were quenched by heating to 95 °C for 5 minutes, followed by the addition of 1 reaction volume of methanol. The protein was then pelleted by centrifugation and the supernatant was analysed by analytical RP- HPLC and LC-HRMS. Assays were analysed on a Shimadzu Analytical UHPLC with a 5 Kinetex 5 μm XB-C18100 x 4.6 mm (Phenomenex) column, flow rate 1 mL / min with a solvent system of H2O (A) / MeOH(B) / 0.1% FA. A gradient of %B from 60-78% was run over 12 min. Heptaenes were monitored at 405 nm and pentaenes were monitored at 350 or 320 nm. LC-HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility Q-TOF LC / MS instrument, using a Luna Omega 5 μm 100 x 2.1 mm 10 (Phenomenex) column, flow rate 1 mL / min with a gradient of 60-95% methanol over 15 min. On LC chromatograms, heptaenes were monitored at 405 nm and pentaenes were monitored at 350 or 320 nm. Scaled up production of polyene derivatives from enzymatic reactions. For 15 scaled up production of glucosylated AmB and Nys, reactions were set up in 1 ml volumes consisting of 5 μM, 2 mM TDP-D-glucose, 2 mM MgCl2, 1 mM polyene in DMSO (10% final DMSO concentration), 50 mM Tris pH 7.5, with at least 101 ml reactions per batch. Reactions were incubated at 30 °C for 18 hrs and were subsequently quenched by heating at 65 °C for 5 min followed by the addition of 1 vol of 20 methanol. Quenched reactions were pooled and centrifuged to remove precipitated protein. The clear supernatant was evaporated in vacuo to dryness. Dry material was dissolved in 1-5 ml DMSO and semi-preparative RP-HPLC (with a gradient of 60%-95% methanol over 30 min) was performed to obtain pure AmB-glc and nys-glc. 25 Expression of KasSub in S. nodosus::KasSub. The sugar subcluster from S. kasugaensis, KasSub, was amplified from S. kasugaensis genomic DNA as three fragments, using primers: L-Fwd (GAGCAACGGAGGTACGGACATATGCATCGAGCGGGTCATCGCC); 30 [SEQ ID NO: 63] L-Rev (CTGCGCGAGCGGTGACCTGCGATGCCCG); [SEQ ID NO: 64] 35 M-Fwd (GCGGTGACCTGCGATGCCCGTCCGCGAG); [SEQ ID NO: 65] 126210PCT1 - 128 - M-Rev (CGAGTCCGCTGAGGGGTCGATATGAAGC); [SEQ ID NO: 66] 5 R-Fwd (CTGAGGGGTCGATATGAAGCTGTCGGAGCGCGCGGAC); [SEQ ID NO: 67] R-Rev (GCCGCCGGCTGACGCCCCGAATTCGTAATCATGTCATAGCTGT). [SEQ ID NO: 68] 10 The three fragments were assembled together using HiFi assembly with pSET152- ermE*, pre-digested with NdeI and EcoRI. The resultant construct, pSET-ermE*- KasSub, was introduced into S. nodosus by standard conjugation protocols, resulting in the S. nodosus::KasSub strain.2215 Production and isolation of polyenes from S. Kasugaensis and S. nodosus::KasSub. Starter cultures were grown in TSB for two days and were used to inoculate 200 mL FSM (fructose 20 g / L, dextrin 60 g / L, soya flour 30 g / L, CaCO310 g / L) fermentation cultures in 1L flasks (2% v / v inoculum). Fermentation cultures were 20 supplemented with 50 g / L amberite XAD16N resin to aid natural product recovery, and were incubated for 7 days, shaking at 30 °C. Mycelia and XAD16 resin were pelleted by centrifugation and two methanol extracts (each one pellet volume) were performed. Extracts were then dried in vacuo to complete or near-complete dryness.50 mL water was added to the dry material, the resulting suspension incubated at 4 °C for 3 hrs, and 25 then centrifuged to produce pellets containing large amounts of polyenes. Polyene pellets were then dissolved in DMSO for purification by semi-preparative RP-HPLC (Shimadzu Prominence HPLC with a Phenomenex Gemini C18 column 250 x 10 mm, 5 μm particle size). Kasufungin B from S. kasugaensis was purified using a 65% isocratic flow of methanol for 11 min followed by a gradient of 66%-84% methanol over 12.5 min. 30 AmB-deoxysugar from S. nodosus::KasSub was purified using a gradient of 70%-88% methanol over 30 min. PcsA Expression and Purification. The E. coli BL21 (DE3) transformed with PcsA- Tag1-pET28a was added into 10 ml LB overnight and was used as starter culture.2xYT 35 medium (1200 mL) was inoculated with 2% of the starter culture with the addition of 50 μg / ml of kanamycin and kept at 37 °C until OD6000.5-0.6 was reached. This was 126210PCT1 - 129 - followed by IPTG induction (0.5 mM) at 18 °C, 180 rpm overnight. After 18 hrs, cultures were centrifuged at 5000 rpm for 10 min, the cell pellets resuspended in ice cold lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 10 mM imidazole, 10 % glycerol, pH 7) and sonicated (Bandelian Sonoplus) on ice for 3 cycles at 70 W power for 8 min 5 times 8 on ice. The samples were centrifuged (10,000 rpm, 45 min), the resultant supernatant was mixed with Ni-NTA agarose (Qiagen) resin and incubated at 4 °C for 45 min. The protein bound resin was first washed with 40 mM imidazole buffer and eluted in 300 mM imidazole buffer (2 mL elution). 10 PcsA Optimized Assay Conditions. Enzyme activity assays were carried out in a reaction mixture containing 100 μM polyene, 2 mM L-Gln / L-glutamic acid γ- monohydroxamate 8 mM ATP, 10 mM MgCl2 and 25 μL of protein elution in a buffer containing 125 mM Tris and 25 mM NaCl (pH 7) in a 50 μL reaction volume. The reactions were quenched with 1 x reaction vol. of methanol after overnight incubation 15 at 30 °C, and centrifuged (10,000 rpm for 5 min). The supernatant was analysed by HPLC and masses were confirmed by LCMS. Analytical RP-HPLC and LC-HRMS Analysis. Polyene extracts and reaction assays were analyzed using a Shimadzu Analytical UHPLC with Phenomenex reverse 20 phase C18, 2.6μ (100 x 4.6 mm) column. The flow rate of 1 mL / min was used. The mobile phase consisted of a mixture of water and methanol (0.1% formic acid each) and a gradient of 65-95% methanol was used over 30 min run time. Tetraenes (Pim, PimNH2, PimNHOH NysNH2, Nys1056 and Nys1056NH2) were monitored at 304 nm. LC-HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility 25 QTOF LC / MS instrument, using a Luna Omega 5 μm 100 x 2.1 mm (Phenomenex) column, flow rate 1 mL / min with a gradient of 60-95% methanol over 15 min. Semi-preparative RP-HPLC. Polyene purification was done using Shimadzu Prominence HPLC with a Phenomenex Gemini C18 column 250 x 10 mm, 5 μm particle 30 size, at 5 mL / min flow rate. Water was used as solvent A and methanol as solvent B without acid addition. A gradient of 40%-55% methanol over 30 min was used for the purification of PimNH2 and PimNHOH while a gradient of 40%-85% methanol over 30 min was used for the purification of NysNH2, Nys1056 and Nys1056NH2. 35 S. albulus Fermentation Conditions. S. albulus (DSM40492) was obtained from DSMZ. Seed culture was maintained in TSB for 2-3 days. SFM agar used for sporulation 126210PCT1 - 130 - and conjugation. FSM medium (Soya flour 30 g, fructose 20 g, dextrin 60 g, CaCO310 g) was used for fermentation (28 °C, 5-7 days). Polyene Purification. To assist polyene (Nys1056) recovery, fermentation cultures 5 were supplemented with amberlite XAD16N resin of 20 g / L in FSM medium and kept for 5-7 days at 30 °C, 180 rpm. Centrifugation (10,000 rpm, 10 min) was performed to obtain the mycelial pellet and polyene bound resin. Solvent extraction was done with 2 x culture volume of methanol and the extracts were dried in vacuo for methanol removal. The water fraction containing polyenes was centrifuged to obtain polyene 10 pellets, dissolved in DMSO and separated on semi-preparative RP-HPLC. The HPLC fractions were dried in vacuo for methanol removal and lyophilized after addition of water. The purified dry polyene powder was used for different tests. Antifungal activity tests for PcsA catalysed Polyene derivatives. For MIC 15 determination, all the strains used were cultured on SAB agar for 3 days at 37 °C. Conidia were collected in PBS supplemented with 0.1% tween-20 (PBST) filtered by autoclaved funnels with sterile Miracloth (Millipore Limited, UK) connected, and followed by quantification with a haemocytometer. MIC of the sample compounds was performed as illustrated in Mendive-Tapia et al., 2017, with trivial changes. Individual 20 compounds were dissolved in DMSO at a concentration of 10 mg / mL and used as the stock solution. For MIC examination, RPMI media was used to dilute the stock solutions to attain a 100 mg / L concentration, followed by the addition to a 96-well cell culture plate. Within the 96-well plate, a serial dilution was prepared. The solutions of compounds at various concentrations were then mixed with a suspension of conidia in 25 RPMI attaining the final volume of 100 μL per well. The resulting concentration of conidia was 5 × 105 cells / mL in RPMI. The maximum confirmed concentration of individual compound was at 50 mg / L. Brightfield microscopy was performed to determine the MIC after incubation of 48 h at 37 °C. 30 Hemolytic tests. Polyene dilutions ranging from (0.01-1 mg / mL) from stocks (4 mg / mL) were prepared in DMSO. From each of the dilution 12 μL was added to 108 μl of PBS buffer containing 2.5% horse blood (Thermofisher) incubated at 37 °C for 1 hr, centrifuged (14,000 rpm for 5 min) and OD545 of the supernatant was measured. A 100% hemolysis was determined by the addition of 12 μL DMSO in distilled water 35 containing 2.5% horse blood. All test compounds were tested in parallel with the 126210PCT1 - 131 - standards in triplicates and the concentrations giving 100% blood hemolysis were recorded. Solubility Test. Nys is soluble in water at 0.1 mg / mL.53 Fifteen times concentrated 5 saturated solutions (1.5 mg / mL) of Nys A1 and Nys1056 were prepared in methanol. A 100 μl portion of each stock was dried under vacuum for 2 h. Aqueous 10 mM Tris-HCl buffer (pH 7.0) was added to the dry polyenes at room temp (20 °C), and the solution was saturated by sonication (15 min) followed by vortexing (30 sec). After centrifugation (10 min at 14,000 g), undissolved polyene pelleted and concentrations in 10 the supernatant were determined by measuring UV absorbance at 304 nm, quantified using standard curve. The assays were repeated in triplicates. Methods for reverse glycosylation and addition of alternative sugars onto polyene aglycones. 15 The reverse glycosylation reactions were performed using crude lysates of the relevant enzymes (MycDI and GDPMH). Two 400 mL cultures of E. coli expressing MycDI and GDPMH separately, were harvested by centrifugation. The resulting cell pellet was resuspended in 25 mL of lysis buffer 50mM Tris-HCL pH 7.0 and 150 mM NaCl and lysed by sonication. The reaction was carried out in a 100 µL volume. The final 20 concentrations of assay components were 200 µM of polyene AmB (used 2 µL of 5 mM stock), 2 mM of guanosine diphosphate (used 2 µL of 100 mM stock) and 48 µL of each enzyme lysate (MycDI and GDPMH), incubated at 30 °C for 18 hrs. The reaction was quenched with an equal volume of methanol and centrifuged (10, 000 rpm, 10 min). The supernatant was analysed for the formation of AmB aglycone by HPLC and 25 chromatograms monitored at 405 nm. For the addition of C-19-mannose onto AmB aglycone, the above reaction containing aglycone was treated (lyophilized, resuspended in methanol, dried and dissolved in 3 µL DMSO) to obtain polyene substrate for the forward reaction. This was incubated 30 with 48 µL of MycDI lysate and 2 mM final GDP-mannose concentration in a 50 µL reaction at 30 °C for 18 hrs. The reaction was quenched with an equal volume of methanol and centrifuged (10, 000 rpm, 10 min). The supernatant was analysed for the formation of AmB -19-man by HPLC and chromatograms monitored at 405 nm. 35 For the addition of C-35 glucose onto AmB-19-man, the above reaction was treated (lyophilized, resuspended in methanol, dried and dissolved in 3 µL DMSO) to obtain 126210PCT1 - 132 - polyene substrate which was then incubated with 15 to 20 µM of purified KfuSV, 2 mM TDP-glucose, 2mM MgCl2 and 50mM Tris-HCL, pH 7.2 in a 50 µL reaction at 30 °C for 18 hrs. The reaction was quenched with an equal volume of methanol and centrifuged (10, 000 rpm, 10 min). The supernatant was analysed for the formation of AmB -19- 5 man-35-glu by HPLC and chromatograms monitored at 405 nm. Methods for determination of novel polyene bioactivity. Antifungal susceptibility testing was performed using the EUCAST (European Committee for Antimicrobial Susceptibility Testing) reference microdilution method 10 version 9.3.2 (73) and 7.4 (74). Yeasts (C. albicans ATCC 90028, C. glabrata NCPF3309, C. auris H.17.157 and Cryptococcus neoformans F10025) were cultured on Sabouraud’s dextrose agar (SDA) plates at 30 ºC for 3 days. Inocula were prepared by suspending five distinct colonies in PBS-0.1% tween and adjusted to 5x105cells / ml in sterile distilled water. Filamentous fungi (Aspergillus fumigatus A1160, Aspergillus 15 fumigatus TR34 / L98H, Rhizopus delemar 99-880, Mucor circinelloides 1006Phl, Fusarium oxysporum 9935, Fusarium solani 9596) were cultured in Sabouraud’s dextrose agar (SDA) T25 vented flasks at 37 ºC for 3 days, except Fusarium species that were cultured at 28 ºC for 5 days. Conidia were harvested with PBS-0.1% tween and filtered through miracloth. Inocula were adjusted to 5x105cells / ml in sterile distilled 20 water. Tests were performed in flat-bottom 96-well plates (CytoOne, StarLab, Belgium) containing 100 μL of 2 x RPMI-1640 medium of a two-fold dilution series of antifungal agents and a drug-free control well. Compound stock solutions were prepared in DMSO and concentrations tested were 12.5 to 0.012 μg / ml for AmB series and 50-0.048 μg / ml for Pim, NysA1 and NysA3 series. Each well was inoculated with 100 μL of the 25 respective strain at a final concentration of 5 x 104conidia. Additionally, Milli-Q water (100 μL) was added to a row of wells in each plate as a sterility control. All plates were incubated at 37°C for 48 h and optical density was measured using a BioTek Synergy 2 SL microplate reader (OD600). From the optical density measurement, the half-maximal inhibitory concentration (IC50) of each drug was determined by fitting inhibitory dose- 30 response curves using variable slope model on GraphPad Prism. Measurement of polyene haemolytic activity on horse blood was performed with slight modifications as described (75). Polyene dilutions ranging from (0.04-350 µM) were prepared from 4 mM stock in DMSO. From each of the dilutions, 6 µL was added to 114 35 µl of PBS buffer containing 2.5% horse blood (Thermofisher) and incubated at 37 °C for 1 hour in 96 well plates, centrifuged (4,000 rpm for 15 min), and OD545of the 126210PCT1 - 133 - supernatant (100 µl) was measured using a plate reader. A total of 16 different final concentrations (0.04, 0.08, 0.3, 1.25, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 µM) were tested for AmB, AmBS, NysA3, Nys31, Nys32, Nys33 and Nys34, 15 final concentrations (0.6, 1.3, 2.5, 10, 30, 50, 70, 100, 120, 140, 160, 200, 250, 300, 350 µM) 5 for NysA1, Nys11, Nys12, Nys13, Nys14 and 13 final concentrations (10, 30, 50, 70, 90 and between 110 to 350 µM) for Pim, Pim1, Pim2, Pim3 and Pim4. Positive and negative controls were determined by adding 6 µL DMSO into 114 µL of 2.5% horse blood in distilled water and PBS buffer, respectively, incubated at 37 °C for 1 hour and OD545was determined as described above. All modified polyenes and standards were 10 tested in parallel with controls in triplicates. The values for 50% horse blood haemolysis (EC50) were determined by a four-parameter logistic curve (4PL) using GraphPad Prism, version 10.2.3 (347). 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Claims

126210PCT1 - 136 - Claims 1. A polyene, wherein the polyene comprises two sugar moieties and / or is modified in an exocyclic carboxylic acid position, or a pharmaceutically acceptable salt, 5 solvate, tautomeric form or polymorphic form thereof, wherein the polyene is not: ,10 2. The polyene of claim 1, wherein the polyene is a compound of formula I:126210PCT1 - 137 -wherein L1is an optionally substituted C2-15alkylene or an optionally substituted C2-155 alkenylene; L2is absent or is an optionally substituted C1-8 alkylene or an optionally substituted C2-8 alkenylene;L3is absent or is , where an asterisk indicates a point of bonding to the carbon atom to which the R1group is bonded; 10 R1is H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl or an optionally substituted C3-12 cycloalkenyl; R2is H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl or an 15 optionally substituted C3-12 cycloalkenyl; R3is an optionally substituted C1-16 alkyl, an optionally substituted C2-16 alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12cycloalkenyl, COOR5, CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor CONR5OOR5a; 20 R4is an optionally substituted sugar moiety; R5, R5aand R5bare each independently H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C3-12 cycloalkenyl, COR8, CONHR8, a C6-12 aryl or a 5 to 10 membered heteroaryl; 25 R6is H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12 cycloalkenyl, an optionally substituted C6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl; R7is OH or an optionally substituted sugar moiety; and126210PCT1 - 138 - R8is H, an optionally substituted C1-16alkyl, an optionally substituted C2-16alkenyl, an optionally substituted C2-16 alkynyl, an optionally substituted C3-12 cycloalkyl or an optionally substituted C3-12 cycloalkenyl; wherein if R3is an optionally substituted C1-16 alkyl, an optionally substituted C2-16 5 alkenyl, an optionally substituted C2-16alkynyl, an optionally substituted C3-12cycloalkyl, an optionally substituted C3-12 cycloalkenyl or COOH then L3is present and R7is an optionally substituted sugar moiety; or a pharmaceutically acceptable salt or solvate thereof; wherein the compound of formula I is not: 10 ,153. The polyene of claim 2, wherein L1is an optionally substituted C3-12 alkylene or an optionally substituted C3-12alkenylene, preferably L1is an optionally substituted C4-10126210PCT1 - 139 - alkylene or an optionally substituted C4-10alkenylene, and most preferably L1is an optionally substituted C5-9 alkylene or an optionally substituted C5-9 alkenylene.

4. The polyene of claim 2 or claim 3, wherein L1is a substituted alkylene or a 5 substituted alkenylene, and the alkylene or alkenylene group is substituted with one or more of OH, -O-, oxo and / or a C1-6 alkyl group.

5. The polyene of any one of claims 2 to 4, wherein L2is absent or is a C1-8 alkylene or a C2-8alkenylene, preferably L2is a C2-6alkylene or a C2-6alkenylene, and more 10 preferably L2is a C2-6 alkenylene.

6. The polyene of any one of claims 2 to 5, wherein L3is absent.

7. The polyene of any one of claims 2 to 5, wherein L3is , and preferably is 15.

8. The polyene of any one of claims 2 to 7, wherein: - R1is H or an optionally substituted C1-12alkyl, an optionally substituted C2-12alkenyl or an optionally substituted C2-12alkynyl; and / or 20 - R2is H, an optionally substituted C1-12 alkyl, an optionally substituted C2-12 alkenyl or an optionally substituted C2-12 alkynyl.

9. The polyene of any one of claims 2 to 8, wherein R3is COOR5, CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor CONR5OOR5a, preferably wherein R5,25R5aand R5bare each be H, a C1-6 alkyl or phenyl.

10. The polyene of any one of claims 2 to 9, wherein R4is a monosaccharide, a disaccharide, a polysaccharide or a derivative thereof. 30 11. The polyene of any one of claims 2 to 10, wherein R6is H or an optionally substituted C1-12alkyl, an optionally substituted C2-12alkenyl or an optionally substituted C2-12 alkynyl.126210PCT1 - 140 - 12. The polyene of any one of claims 2 to 11, wherein R7is a monosaccharide, a disaccharide, a polysaccaride or a derivative thereof. 5 13. The polyene of claim 1 or claim 2, wherein the polyene is , , , , 10,126210PCT1 - 142 - , , ,,126210PCT1 - 145 - ,,126210PCT1 - 146 - ,,126210PCT1 - 147 -126210PCT1 - 148 -126210PCT1 - 149 -.

14. A pharmaceutical composition comprising the polyene of any one of the 5 preceding claims, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, and a pharmaceutically acceptable vehicle.

15. The polyene of any one of claims 1 to 13, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, or the 10 pharmaceutical composition of claim 14, for use as a medicament.

16. The polyene of any one of claims 1 to 13, or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof, or the pharmaceutical composition of claim 14, for use in treating, preventing or ameliorating an infection or a 15 prion disease.

17. The polyene or composition for use according to claim 16, wherein the infection is a microbial infection, and preferably is a fungal infection or a parasitic infection. 20 18. The polyene or composition for use according to claim 17, wherein the microbial infection is a fungal infection caused by a fungus in the genus Aspergillus, Candida, Coccidioides, Cryptococcus, Histoplasma, Fusarium, Scedosporium or Trichosporon.

19. An agrochemical composition comprising the polyene of any one of claims 1 to25126210PCT1 - 150 - 20. Use of the polyene of any one of claims 1 to 13 or the agrochemical composition of claim 19 to treat a disease in a plant or a mushroom.

21. A method of treating a disease in a plant or a mushroom, the method 5 comprising contacting a plant or mushroom in need of such a treatment with the polyene of any one of claims 1 to 13 or the agrochemical composition of claim 19.

22. A preservative comprising the polyene any one of claims 1 to 13. 10 23. A foodstuff, comprising the polyene of any one of claims 1 to 13 or the preservative of claim 22.

24. Use of the polyene of any one of claims 1 to 13 or the food preservative of claim 22 to preserve a foodstuff. 15 25. A method of producing a polyene comprising a sugar moiety, the method comprising contacting a polyene with a glycosyltransferase enzyme in the presence of a nucleotide-linked sugar to thereby produce the polyene comprising a sugar moiety. 20 26. The method of claim 25, wherein the polyene which is contacted with the glycosyltransferase enzyme in the presence of a nucleotide-linked sugar, is a polyene of formula VIII:25 and the polyene which is produced is a polyene of formula IX:126210PCT1 - 151 -wherein, R1to R4, R6, L1and L2are as defined in any one of claims 2 to 13; and 5 R7ais a sugar moiety.

27. The method of claim 25 or claim 26, wherein prior to contacting the polyene with the glycosyltransferase enzyme in the presence of the nucleotide-linked sugar to produce the polyene comprising a sugar moiety, the method comprises: 10 - contacting a polyene comprising a sugar moiety with a glycosyltransferase enzyme in the presence of a nucleoside diphosphate (NDP)-sugar hydrolase, to thereby remove the sugar moiety from the polyene.

28. A method of producing a polyene comprising a modification at an exocyclic 15 carboxylic acid thereon, the method comprising contacting the polyene with an amidotransferase enzyme in the presence of a nitrogen donor to thereby produce the modified polyene, wherein the polyene which is contacted with the amidotransferase is 20and the polyene which is produced is a polyene of formula XI:126210PCT1 - 152 -wherein, R1, R2, R4, R5, R5a, R5band L1to L3are as defined in any one of claims 2 to 13; 5 and R3ais CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor CONR5OOR5a.

29. A method of producing a modified polyene comprising a sugar moiety and comprising a modification at an exocyclic carboxylic acid thereon, the method 10 comprising: - contacting a polyene and a glycosyltransferase enzyme in the presence of a nucleotide-linked sugar; and - contacting the polyene and an amidotransferase enzyme in the presence of a nitrogen donor; 15 to thereby produce the modified polyene, wherein prior to contacting either the glycosyltransferase enzyme or the amidotransferase enzyme, the polyene is a compound of formula XIII:20 and the method produces a polyene of formula XIV:126210PCT1 - 153 -wherein R7ais a sugar moiety; 5 R3ais CONR5R5a, CONR5NR5aR5b, CONR5OR5a, CONR5NR5aOR5bor CONR5OOR5a; and R1, R2, R4to R6, L1and L2are as defined in any one of claims 2 to 13.

30. The method of any one of claims 28 to 29, wherein the amidotransferase enzyme is a Class II glutamine amidotransferase (GAT) enzyme. 10 31. The method of any one of claims 28 to 30, wherein the amino acid sequence of the amidotransferase enzyme comprises at least 30% sequence identity, at least 40% sequence identity or at least 50% sequence identity, preferably at least 60% sequence identity, more preferably at least 70% sequence identity, to the sequence identified as 15 SEQ ID NO: 16 or SEQ ID NO:

17.

32. The method of any one of claims 28 to 31, wherein the nitrogen donor is ammonia, an ammonium ion, an ammonium salt or a compound of formula XII: 20wherein R14is NR5R5a, NR5NR5aR5b, NR5OR5a, NR5NR5aOR5b, NR5OOR5aor NR5CONR5aR5b. 25 33. The method of any one of claims 25 to 27 or 29 to 32, wherein the glycosyltransferase enzyme comprises at least 30% sequence identity, at least 40% sequence identity or at least 50% sequence identity to any one of SEQ ID Nos: 1-6, 30, 72 and 73, and preferably has at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to any one of SEQ ID Nos: 1-6, 30, 72 and 73.126210PCT1 - 154 - 34. A recombinant organism or cell expressing a glycosyltransferase enzyme comprising at least 30% sequence identity, at least 40% sequence identity or at least 50% sequence identity to any one of SEQ ID Nos: 1-6, 30, 72 and 73, and preferably has 5 at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to any one of SEQ ID Nos: 1-6, 30, 72 and 73.

35. A recombinant organism or cell expressing an amidotransferase enzyme comprising at least 30% sequence identity, at least 40% sequence identity or at least 10 50% sequence identity, preferably at least 60% sequence identity, more preferably at least 70% sequence identity, to the sequence identified as SEQ ID NO: 16 or SEQ ID NO:

17.

36. The recombinant organism or cell of claim 35, wherein the recombinant 15 organism also expresses a glycosyltransferase enzyme comprising at least 30% sequence identity, at least 40% sequence identity or at least 50% sequence identity to any one of SEQ ID Nos: 1-6, and preferably has at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to any one of SEQ ID Nos: 1-6.

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