Psilocybin, precursors and derivatives thereof

Abstract

The present disclosure relates to psilocybin, intermediates of psilocybin, derivatives of psilocybin and to methods of producing psilocybin, intermediates and derivatives thereof. In some aspects, the invention uses cells engineered to express enzymes.

Description

PSILOCYBIN, PRECURSORS AND DERIVATIVES THEREOFField of the disclosure

[0001] The present disclosure relates to psilocybin, intermediates of psilocybin, derivatives of psilocybin and to methods of producing psilocybin, intermediates and derivatives thereof.Incorporation by reference

[0002] The present application claims priority from Australian provisional application no. 2024903091 , the entire contents of which are incorporated herein by reference.Background of the disclosure

[0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0004] Psilocybin, the phosphorylated form of the psychoactive alkaloid, psilocin, is mainly found in the genus Psilocybe, colloquially known as magic mushrooms (Figure 1). Psilocybin has been seen as a promising candidate for the treatment of a broad range of mental health-related conditions and neurological disorders. The Australian Therapeutic Goods Administration has approved the use of psilocybin for treatment-resistant depression.

[0005] The mode of action of psilocin, which is cleaved from psilocybin after ingestion, is inferred by its agonistic effect on the 5-hydroxytryptamine receptors, 5-HT2A and 5-HT2C (Fricke, J., et al., (2019) Journal of Natural Products 25, 897 - 903).

[0006] In nature, psilocybin is found in a large variety of Psilocybe and other genera of mushrooms (McKernan, K., et al., A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production. F1000Research 2021 , 10.961). The psilocybin content in mushrooms varies between 0.2% and 1.7% (Mahmood, Z. A., Chapter 18, Bioactive alkaloids from fungi: psilocybin, In Natural Products, Eds: Ramawat, K. G. and Merillon, J. M. (2013) Springer Verlag Berlin Heidelberg, DOI: 10.1007 / 978-3- 642-22144-6_19). Commercial production of psilocybin in mushrooms is hampered by the low and variable content of psilocybin, the coupling of psilocybin production to the slow growth cycle, and intracellular psilocybin production requiring psilocybin purification from a complex matrix, adding to process cost and decreasing yield.

[0007] Psilocybin for clinical trials is currently produced via chemical synthesis (Fricke, J., et al., (2019) Journal of Natural Products 25, 897 - 903). Based on the original Hoffmann synthesis, Fricke et al., estimated the cost of producing 1 gram of psilocybin for phase III clinical studies was more than US$2000 (Fricke, J. et al., (2020) Chemistry 26(37), 8281 - 8285; 10. Fricke, J., et al., (2017) Angewandte Chemie International Edition 56, 12352 - 12355). However, the chemical syntheses are associated with low atom efficiency especially for the phosphorylation steps, multiple solvents, and an inefficient product isolation process generating a large amount of waste (Sherwood, A. M., et al., (2020) Synthesis 52, 688 - 694). A hybrid approach has been developed involving the chemical synthesis of psilocin followed by enzymatic phosphorylation of psilocin producing psilocybin. However, the process requires the purification of psilocin prior to the kinase step, which increases the number of unit operations, impacts yield, and increases waste production (Fricke, J. et al., (2020) Chemistry 26(37), 8281 - 8285; 10).AU_Active01 27520873v1 CHRISTM

[0008] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.Summary of the disclosure

[0009] In work leading to the present disclosure, the inventors sought to produce psilocybin, precursors of psilocybin and derivatives of psilocybin.

[0010] In one aspect, the present disclosure provides a method of producing psilocybin comprising:(a) preparing a reaction mixture comprising tryptamine, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase, a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4- hydroxytryptamine, the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin, the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

[0011] Where the present disclosure refers to the presence of a strain of cells or a lysate thereof, it will be understood that the strain of cells may be present (ie, as whole cells), or a lysate of that strain may be present, or both the cells and the lysate may be present. For example, in the aspect immediately above, the reaction mixture may comprise the first strain of cells (ie, as whole cells) and a lysate of the second strain of cells and a lysate of the third strain of cells. Or, in some examples, the reaction mixture comprises the second strain of cells (ie, as whole cells) and a lysate of the first strain of cells and a lysate of the third strain of cells. In some examples, the reaction mixture comprises the third strain of cells (ie, as whole cells) and a lysate of the first strain of cells and a lysate of the second strain of cells. In some examples, the reaction mixture comprises the first strain of cells, the second strain of cells and a lysate of the third strain of cells. In some examples, the reaction mixture comprises the second strain of cells and the third strain of cells and a lysate of the first strain of cells. In some examples, the reaction mixture comprises the first strain of cells and the third strain of cells and a lysate of the second strain of cells. In some examples, the reaction mixture comprises the first strain of cells, the second strain of cells and the third strain of cells; that is, step (a) of the method may comprise preparing a reaction mixture comprising: tryptamine, a first strain of cells wherein the first strain is engineered to express a monooxygenase, a second strain of cells wherein the second strain is engineered to express a kinase, and a third strain of cells wherein the third strain is engineered to express a methyltransferase.AU_Active01 27520873v1 CHRISTM

[0012] In some examples, the monooxygenase is a cytochrome P450 monooxygenase. It will be understood that the cytochrome P450 monooxygenase described herein is capable of hydroxylating tryptamine to produce 4-hydroxytryptamine, ie, the cytochrome P450 monooxygenase is a tryptamine 4- hydroxylase. In some examples, the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof. In some examples, the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

[0013] In some examples, the first strain is engineered to express a reductase. The reductase may be a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof. In some examples, the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3. In some examples, the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.

[0014] In some examples: the monooxygenase and / orthe reductase are encoded by nucleic acids codon optimised for expression in the first strain; the kinase is encoded by a nucleic acid codon optimised for expression in the second strain; and / or the methyltransferase is encoded by a nucleic acid codon optimised for expression in the third strain.

[0015] In some examples, the first strain of cells has been cultured in the presence of b-aminolevulinic acid. In some examples, the first strain of cells has been cultured in the absence of b-aminolevulinic acid. In some examples, the first strain of cells has been cultured in a medium comprising soytone.

[0016] In some examples, the kinase is a Psilocybe cubensis 4-hydroxytryptamine kinase or a variant or derivative thereof. The kinase may comprise the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.

[0017] In some examples, the methyltransferase is a / V-methyltransferase. In some examples, the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof. In some examples, the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.

[0018] In some examples, the reaction mixture further comprises S-adenosylmethionine (SAM) which provides a methyl for the methylation reactions. In some examples, the reaction mixture further comprises adenosine triphosphate. In some examples, the reaction mixture further comprises methionine. In some examples, the reaction mixture further comprises L-methionine. In some examples, the reaction mixture further comprises glycerol or glucose. In some examples, the reaction mixture further comprises a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate.

[0019] In some examples, the reaction mixture comprises intact cells of the first strain, intact cells of the second strain and / or intact cells of the third strain. In some examples, the first strain, the second strain and / or the third strain carry a defective or suppressed tryptophanase.

[0020] In some examples, the reaction mixture further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.AU_Active01 27520873v1 CHRISTM

[0021] In some examples, the first strain, the second strain and the third strain are different strains of microorganisms. In some examples, the first strain, the second strain and the third strain are different strains of E. coli.

[0022] In some examples, preparing the reaction mixture comprises separately adding the first strain, the second strain and the third strain to the reaction mixture. In some examples, the first strain, the second strain and the third strain are separately cultured before each strain is added to the reaction mixture.

[0023] In some examples, the second strain does not comprise: a Psilocybe cubensis methyltransferase or a variant or derivative thereof; and / or a tryptophan decarboxylase. In some examples, the third strain does not comprise: a 4-hydroxytryptamine kinase; and / or a tryptophan decarboxylase.

[0024] In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells has been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells has been spray dried, freeze dried, air dried or vacuum dried. In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells has been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol and preferably magnesium sulfate.

[0025] In a second aspect, the present disclosure provides a method of producing 4-hydroxytryptamine comprising: preparing a reaction mixture comprising tryptamine, and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase; and incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine.

[0026] In some examples, the monooxygenase is a cytochrome P450 monooxygenase. In some examples, the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof. In some examples, the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

[0027] In some examples, the cells are engineered to express a reductase. In some examples, the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof. In some examples, the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3. In some examples, the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites. In some examples, the monooxygenase and the reductase are encoded by a nucleic acid codon optimised for expression in the cells.

[0028] In some examples, the reaction mixture comprises intact cells. In some examples, the cells carry a defective or suppressed tryptophanase.

[0029] In some examples, the reaction mixture further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.AU_Active01 27520873v1 CHRISTM

[0030] In some examples, the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol and preferably magnesium sulfate.

[0031] In a third aspect, the present disclosure provides a method of producing norbaeocystin comprising: preparing a reaction mixture comprising4-hydroxytryptamine, and cells or a lysate thereof wherein the cells are engineered to express a kinase; and incubating the reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin

[0032] In some examples, the kinase is a 4-hydroxytryptamine kinase. In some examples, the kinase is a Psilocybe cubensis 4-hydroxytryptamine kinase or a variant or derivative thereof. In some examples, the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4. In some examples, the kinase is encoded by a nucleic acid codon optimised for expression in the cells.

[0033] In some examples, the reaction mixture further comprises adenosine triphosphate. In some examples, the reaction mixture further comprises glycerol or glucose. In some examples, the reaction mixture further comprises a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate.

[0034] In some examples, the reaction mixture comprises intact cells. In some examples, the cells carry a defective or suppressed tryptophanase.

[0035] In some examples, the reaction mixture further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

[0036] In some examples, the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol and preferably magnesium sulfate.

[0037] In some examples, the reaction mixture further comprises tryptamine, and cells engineered to express a monooxygenase and a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine to produce the 4- hydroxytryptamine; and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.

[0038] In some examples, the 4-hydroxytryptamine is produced by any one of the methods described in the second aspect.AU_Active01 27520873v1 CHRISTM

[0039] In a fourth aspect, the present disclosure provides a method of producing psilocybin comprising: preparing a reaction mixture comprising norbaeocystin, and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase; and incubating the reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

[0040] In some examples, the methyltransferase is a / V-methyltransferase. In some examples, the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof. In some examples, the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.

[0041] In some examples, the reaction mixture further comprises S-adenosylmethionine which provides a methyl for the methylation reactions. In some examples, the reaction mixture further comprises adenosine triphosphate and L-methionine to facilitate the generation of S-adenosylmethionine.

[0042] In some examples, the methyltransferase is encoded by a nucleic acid codon optimised for expression in the cells.

[0043] In some examples, the reaction mixture comprises intact cells. In some examples, the cells carry a defective or suppressed tryptophanase.

[0044] In some examples, the reaction mixture further comprises methionine. In some examples, the reaction mixture further comprises L-methionine.

[0045] In some examples, the reaction mixture further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

[0046] In some examples, the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol and preferably magnesium sulfate.

[0047] In some examples, the reaction mixture further comprises 4-hydroxytryptamine, and cells engineered to express a kinase or a lysate thereof and wherein the reaction mixture is incubated under conditions suitable for: the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce the norbaeocystin; the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin; and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin

[0048] In some examples, the norbaeocystin is produced by any one of the methods described in the third aspect.AU_Active01 27520873v1 CHRISTM

[0049] In some examples, the reaction mixture further comprises tryptamine, and cells engineered to express a monooxygenase and a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine to produce the 4- hydroxytryptamine; the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin; the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin; and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin

[0050] In some examples, the 4-hydroxytryptamine is produced according to any one of the methods described in second aspect.

[0051] In a fifth aspect, the present disclosure provides a method of producing psilocybin comprising:(a) preparing a first reaction mixture comprising tryptamine, and a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase,(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine;(c) preparing a second reaction mixture by combining the 4-hydroxytryptamine with a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin, the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

[0052] In a sixth aspect, the present disclosure provides a method of producing psilocybin comprising:(a) preparing a first reaction mixture comprising tryptamine, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase, and a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase,(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4- hydroxytryptamine, and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin,AU_Active01 27520873v1 CHRISTM(c) preparing a second reaction mixture by combining the norbaeocystin with a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

[0053] In some examples, the first reaction mixture and / or the second reaction mixture further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

[0054] In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells have been dried or powderised and optionally rehydrated before preparing the first and / or second reaction mixture. In some examples, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

[0055] In a seventh aspect, the present disclosure provides a system for producing psilocybin comprising: tryptamine; a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase; a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase; and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase, wherein: the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4- hydroxytryptamine; the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin; the methyltransferase is capable of catalysing methylation of the norbaeocystin to produce baeocystin; and the methyltransferase is capable of catalysing methylation of the baeocystin to produce psilocybin.

[0056] In some examples, the monooxygenase is a cytochrome P450 monooxygenase. In some examples, the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof. In some examples, the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

[0057] In some examples, the first strain is engineered to express a reductase. In some examples, the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof. In someAU_Active01 27520873v1 CHRISTMexamples, the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3. In some examples, the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites. In some examples: the monooxygenase and / or the reductase are encoded by nucleic acids codon optimised for expression in the first strain; the kinase is encoded by a nucleic acid codon optimised for expression in the second strain; and / or the methyltransferase is encoded by a nucleic acid codon optimised for expression in the third strain.

[0058] In some examples, the first strain of cells has been cultured in the presence of b-aminolevulinic acid. In some examples, the first strain of cells has been cultured in the absence of b-aminolevulinic acid. In some examples, the first strain of cells has been cultured in a medium comprising soytone.

[0059] In some examples, the kinase is a Psilocybe cubensis 4-hydroxytryptamine kinase or a variant or derivative thereof. In some examples, the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.

[0060] In some examples, the methyltransferase is a / V-methyltransferase. In some examples, the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof. In some examples, the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.

[0061] In some examples, the system further comprises S-adenosylmethionine (SAM). In some examples, the system further comprises adenosine triphosphate and L-methionine.

[0062] In some examples, the system comprises intact cells of the first strain, intact cells of the second strain and / or intact cells of the third strain. In some examples, the first strain, the second strain and / or the third strain carry a defective or suppressed tryptophanase.

[0063] In some examples, the system further comprises a membrane permeabilising agent. In some examples, the permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

[0064] In some examples, the first strain, the second strain and the third strain are different strains of microorganisms. In some examples, the first strain, the second strain and the third strain are different strains of E. coll.

[0065] In some examples, the second strain does not comprise: a Psilocybe cubensis methyltransferase or a variant or derivative thereof; and / or a tryptophan decarboxylase. In some examples, the third strain does not comprise: a 4-hydroxytryptamine kinase; and / or a tryptophan decarboxylase.

[0066] In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose or sorbitol, glycerol and preferably magnesium sulfate.AU_Active01 27520873v1 CHRISTM

[0067] In an eighth aspect, the present disclosure provides a system for producing 4-hydroxytryptamine comprising: tryptamine; and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4- hydroxytryptamine.

[0068] In some examples, the monooxygenase is a cytochrome P450 monooxygenase. In some examples, the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof. In some examples, the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

[0069] In some examples, the cells are engineered to express a reductase. In some examples, the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof. In some examples, the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3. In some examples, the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites. In some examples, the monooxygenase and the reductase are encoded by a nucleic acid codon optimised for expression in the cells.

[0070] In some examples, the system comprises intact cells. In some examples, the cells carry a defective or suppressed tryptophanase.

[0071] In some examples, the system further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

[0072] In some examples, the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol and preferably magnesium sulfate.

[0073] In a ninth aspect, the present disclosure provides a system for producing norbaeocystin comprising:4-hydroxytryptamine, and cells or a lysate thereof wherein the cells are engineered to express a kinase, wherein the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.

[0074] In some examples, the kinase is a 4-hydroxytryptamine kinase. In some examples, the kinase is a Psilocybe cubensis 4-hydroxytryptamine kinase or a variant or derivative thereof. In some examples, the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4. In some examples, the kinase is encoded by a nucleic acid codon optimised for expression in the cells.

[0075] In some examples, the system comprises intact cells. In some examples, the cells carry a defective or suppressed tryptophanase.AU_Active01 27520873v1 CHRISTM

[0076] In some examples the system further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

[0077] In some examples, the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

[0078] In some examples, the system further comprises: tryptamine; and cells engineered to express a monooxygenase and a reductase or a lysate thereof, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4-hydroxytryptamine.

[0079] In a tenth aspect, the present disclosure provides a system for producing psilocybin comprising: norbaeocystin; and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase, wherein the methyltransferase is capable of catalysing: methylation of the norbaeocystin to produce baeocystin; and methylation of the baeocystin to produce psilocybin.

[0080] In some examples, the methyltransferase is a / V-methyltransferase. In some examples, the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof. In some examples, the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.

[0081] In some examples, the system further comprises S-adenosylmethionine. In some examples, the system further comprises adenosine triphosphate and L-methionine.

[0082] In some examples, the methyltransferase is encoded by a nucleic acid codon optimised for expression in the cells.

[0083] In some examples, the system further comprises intact cells. In some examples, the cells carry a defective or suppressed tryptophanase.

[0084] In some examples, the system further comprises a membrane permeabilising agent. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

[0085] In some examples, the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

[0086] In some examples, the system further comprises: 4-hydroxytryptamine; and cells engineered to express a kinase or a lysate thereof, wherein the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce the norbaeocystin. In some examples, the system further comprisesAU_Active01 27520873v1 CHRISTMtryptamine; and cells engineered to express a monooxygenase and a reductase or a lysate thereof, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce the 4- hydroxytryptamine.Brief description of the drawings

[0087] Figure 1. Structure of psilocybin.

[0088] Figure 2. Psilocybin biosynthetic pathway in Psilocybe cubensis.

[0089] Figure 3. Effect of different additives on the methyltransferase reaction (PsiM) in whole cells after assaying for 26 hours (negative (-) = negative control without cells or additives; negative (+) = negative control without additive, with cells; Additives: Tween 20 & Triton X-100 = non-ionic detergents, deoxycholate & CTAB = ionic detergents, methanol, iso-propanol & DMSO = polar solvents; deoxycholate = sodium deoxycholate; CTAB = cetyltriammonium bromide; DMSO = dimethylsulfoxide).

[0090] Figure 4. Bioconversion of tryptamine to norbaeocystin (PsiH_trunc_CPR = cytochrome P450 monooxygenase (tryptamine 4-hydroxylase), PsiK = 4-hydroxytryptamine kinase, mM = millimolar, h = hours, TRN = tryptamine, 4OHTRN = 4-hydroxytryptamine, NOR = norbaeocystin).

[0091] Figure 5. One-pot bioconversion of 0.7 g / L (4.4 mM) tryptamine to psilocybin (PsiH_trunc_CPR = cytochrome P450 monooxygenase (tryptamine 4-hydroxylase), PsiK = 4-hydroxytryptamine kinase, PsiM = norbaeocystin methyltransferase, mM = millimolar, h = hours, TRN = tryptamine, 4OHTRN = 4- hydroxytryptamine, NOR = norbaeocystin, BAEO = baeocystin, PBN = psilocybin).

[0092] Figure 6. One-pot bioconversion of 1 .5 g / L (9.4 mM) tryptamine to psilocybin in fermenter (PsiH_trunc_CPR = cytochrome P450 monooxygenase (tryptamine 4-hydroxylase), PsiK = 4- hydroxytryptamine kinase, PsiM = norbaeocystin methyltransferase, mM = millimolar, h = hours, TRN = tryptamine, 4OHTRN = 4-hydroxytryptamine, NOR = norbaeocystin, BAEO = baeocystin, PBN = psilocybin).

[0093] Figure 7. Two-pot bioconversion of 1 .5 g / L (9.4 g / L) tryptamine to psilocybin (PsiH_trunc_CPR = cytochrome P450 monooxygenase (tryptamine 4-hydroxylase), PsiK = 4-hydroxytryptamine kinase, PsiM = norbaeocystin methyltransferase, mM = millimolar, h = hours, TRN = tryptamine, 4OHTRN = 4- hydroxytryptamine, NOR = norbaeocystin, BAEO = baeocystin, PBN = psilocybin).

[0094] Figure 8. Activity of E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8) after freeze-drying without any additives (samples analysed by TLC). Lane 1 : 0-hour sample. Lane 2: 20-hour sample. Lane 3: 48-hour sample.

[0095] Figure 9. Effect of different stabilisers on the activity of cytochrome P450 monooxygenase (PsiH_trunc_CPR) during the freeze-drying process (20-hour samples analysed by TLC and UPLC; cells were rehydrated before assaying the activity of PsiH_trunc_CPR; TRN = tryptamine, 4OHTRN = 4- hydroxytryptamine). Lane 1 : 2% glucose + 25 mM MgSO4 (Glu; 0.62 mM 4OHTRN produced) added to E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells before drying. Lane 2: 2% sucrose + 25 mM MgSCU (Sue; 2.85 mM 4OHTRN produced) added to E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells before drying. Lane 3: 2% sorbitol + 25 mM MgSCU (Sorb; 3.65 mM 4OHTRN produced) added to E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells before drying. Lane 4: 2% trehalose + 25 mM MgSCU (Tre; 1 .88AU_Active01 27520873v1 CHRISTMmM 4OHTRN produced) added to E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells before drying. Lane 5: 2% sucrose + 0.5% glycerol + 25 mM MgSC (+; 2.46 mM 4OHTRN produced) added to E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells before drying.

[0096] Figure 10. Stability of cytochrome P450 monooxygenase (tryptamine 4-hydroxylase) and its cytochrome P450 reductase partner (PsiH_trunc_CPR) after storing the dried E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells at -20°C, 4°C & 25°C (* = samples analysed by UPLC at a later time point, PsiH = PsiH_trunc_CPR = cytochrome P450 monooxygenase (tryptamine 4-hydroxylase) and partner cytochrome P450 reductase, 16 & 20 h = 16- and 20-hour assay samples, 0 & 2 wk = dried and wet cells assayed after 0 and 2 week storage at the respective temperatures, 1 mo = dried and wet cells assayed after 1 month storage at the respective temperatures, mM = millimolar, h = hour).

[0097] Figure 11. Stability of 4-hydroxytryptamine kinase (PsiK) after storing dried E. coli KRXAtnaA / pET29b_PsiK cells at 4°C and 25°C (* starting concentration of 4-hydroxytryptamine in the PsiK assay was 6.81 mM compared to 2.78 mM for the other samples & these samples were analysed at a later time point, 0 and 1 wk = dried and wet cells assayed after 0 and 1 week storage at the respective temperatures, 1 mo = dried and wet cells assayed after 1 month storage at the respective temperatures, 8 h = 8-hour assay sample, mM = millimolar, h = hour).

[0098] Figure 12. Stability of norbaeocystin methyltransferase (PsiM) after storing dried E. coli BL21 (DE3) / pET29b_PsiM cells at -20°C, 4°C & 25°C (* = samples analysed by UPLC at a later time point, 0 and 1 wk = dried and wet cells assayed after 0 and 1 week storage at the respective temperatures, 1 mo = dried and wet cells assayed after 1 month storage at the respective temperatures, 20 and 24 h = 20- and 24-hour assay sample).

[0099] Figure 13. Stability of 4-hydroxytryptamine kinase (PsiK) after storing dried E. coli KRXAtnaA / pET29b_PsiK cells (cryoprotectant = additive = MgSC ) at 4°C & 25°C (PsiK = 4- hydroxytryptamine kinase, 2 h = 2-hour assay samples, 0 & 2 wk = dried cells assayed after 0 and 2 week storage at the respective temperatures, 1 mo = dried cells assayed after 1 month storage at the respective temperatures, mM = millimolar, h = hour).

[0100] Figure 14. Stability of 4-hydroxytryptamine kinase (PsiK) after storing dried E. coli KRXAtnaA / pET29b_PsiK cells (cryoprotectant = additive = MgSC + sorbitol) at 4°C & 25°C (PsiK = 4- hydroxytryptamine kinase, 2 h = 2-hour assay samples, 0 & 2 wk = dried cells assayed after 0 and 2 week storage at the respective temperatures, 1 mo = dried cells assayed after 1 month storage at the respective temperatures, mM = millimolar, h = hour).

[0101] Figure 15. Stability of 4-hydroxytryptamine kinase (PsiK) after storing dried E. coli KRXAtnaA / pET29b_PsiK cells (cryoprotectant = additive = MgSC + trehalose) at 4°C & 25°C (PsiK = 4- hydroxytryptamine kinase, 2 h = 2-hour assay samples, 0 & 2 wk = dried cells assayed after 0 and 2 week storage at the respective temperatures, 1 mo = dried cells assayed after 1 month storage at the respective temperatures, mM = millimolar, h = hour).AU_Active01 27520873v1 CHRISTMDetailed descriptionDefinitions

[0102] In the context of this specification, the terms "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0103] The term "about" is understood to refer to a range of + / - 10%, preferably + / - 5% or + / - 1 % or, more preferably, + / - 0.1 %.

[0104] The terms "comprise", "comprises", "comprised" or "comprising", "including" or "having" and the like in the present specification and claims are used in an inclusive sense, ie, to specify the presence of the stated features but not preclude the presence of additional or further features.

[0105] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences when the sequences are optimally aligned (ie, % homology = # of identical positions / total # of positions x 100), with optimal alignment determined taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0106] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11- 17 (1989)) which has been incorporated into the ALIGN program, using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. Other computer programs that can be used in this regard include BLASTP, BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990:215:403). Another method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, JG, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) using a gap opening penalty of 10 and a gap extension penalty of 0.1 , so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.

[0107] The term “isolated” as used herein refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, an isolated polynucleotide as used herein refers to a polynucleotide which has been purified from the sequences which flank it in a naturally occurring state, eg, a DNA fragment which has been removed from the sequences that areAU_Active01 27520873v1 CHRISTMnormally adjacent to the fragment. Alternatively, an isolated enzyme, as used herein, may refer to in vitro isolation and / or purification of the enzyme from its cellular environment, and from association with other components of the cell, ie, it is not associated with in vivo substances. An isolated enzyme will generally encompass recombinantly expressed enzymes.

[0108] As used herein, the term "operably connected" and its grammatical equivalents refers to the association of nucleic acid sequences on a single polynucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).

[0109] The term "pharmaceutically acceptable" as used herein refers to substances that do not cause substantial adverse allergic or immunological reactions when administered to a subject. A "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, coatings, dispersion agents, wetting agents, isotonic and absorption delaying agents and dis integrants.

[0110] The term “polynucleotide variant” refers to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridise with a reference sequence under stringent conditions. The term also encompasses polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the term “polynucleotide variant” includes polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. The term “polynucleotide variant” also includes naturally occurring allelic variants. The terms “peptide variant”, “polypeptide variant”, “enzyme variant” and the like includes peptides, polypeptides and enzymes that are distinguished from a reference peptide, polypeptide or enzyme by the addition, deletion or substitution of at least one amino acid residue. In certain embodiments, a peptide, polypeptide or enzyme variant is distinguished from a reference peptide, polypeptide or enzyme by one or more substitutions, which may be conservative or non-conservative. In certain examples, the peptide, polypeptide or enzyme variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the peptide, polypeptide or enzyme. Peptide, polypeptide and enzyme variants also encompass peptides, polypeptides and enzymes in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.

[0111] As used herein, the term "recombinant" and its grammatical equivalents means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Thus, as used herein, the terms "recombinant polynucleotide" or "recombinant nucleic acid" refer to a polynucleotide that is non-naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination may be accomplished by chemical synthesis means, orAU_Active01 27520873v1 CHRISTMby the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. A "recombinant cell" is a cell that has been genetically modified or altered to comprise a nucleic acid sequence that is not native to the cell. The engineered cells described herein a preferably recombinant cells that have been genetically modified to express and enzyme.

[0112] The term "substantially complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to, and form a duplex structure with, an oligonucleotide or polynucleotide comprising the second nucleotide sequence. It will be understood that the sequence of a nucleic acid need not be 100% complementary to that of its target. Conditions under which hybridisation occurs may be stringent, such as 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can also apply. Substantial complementarity allows the relevant function of the nucleic acid to proceed. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0113] The term “substantially free” as used herein refers to the indicated component being either not detectable in the reaction mixture by common analytical methods, or being present in such trace amount as to not have a material effect on the reactions taking place in the reaction mixture.As used herein, the term "vector" and its grammatical equivalents refers to any means by which a nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes", that is, that replicate autonomously or can integrate into a chromosome of a host microorganism. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more polynucleotide constructs such as an agrobacterium or a bacterium.

[0114] Where numerical ranges are used to describe certain embodiments of the present disclosure, it will be understood that each range should be considered to encompass subranges therein. For example, the description of a range such as from 1 to 6 should be considered to include subranges such as from 1 to 5, from 2 to 4, from 2 to 6 and so on. Likewise, the description of a range of between 1 and 6 should be considered to include subranges such as between 2 and 5, between 1 and 3, between 3 and 6 and so on.Production of psilocybin and intermediates and derivatives thereof

[0115] The biosynthetic pathway for psilocybin formation in Psilocybe cubensis starting from L- tryptophan was described by Fricke et al. ((2017) Angewandte Chemie International Edition 56, 12352 - 12355) and is illustrated in Figure 2. In the first step, L-tryptophan is decarboxylated by the PLP- independent tryptophan decarboxylase (PsiD) producing tryptamine. Tryptamine is subsequently hydroxylated in 4-position by a cytochrome P450 monooxygenase (PsiH). The product of this reaction,AU_Active01 27520873v1 CHRISTM4-hydroxytryptamine, undergoes a 4-O-phosphorylation by an ATP-dependent 4-hydroxytryptamine kinase (PsiK) producing norbaeocystin. The last two steps of the pathway involve the iterative methylation of norbaeocystin to baeocystin and baeocystin to psilocybin by the S-adenosylmethionine-dependent methyltransferase (PsiM).

[0116] The present disclosure describes inter alia one pot and multi pot biocatalytic approaches with whole cells to synthesise psilocybin, obviating the need for engineering a microbial system with balanced flux through the pathway; uncoupling synthesis from growth affording more process flexibility; optionally avoiding the expensive starting material, 4-hydroxyindole; simplifying the downstream process as the reaction may be carried out in a simple buffer system instead of complex growth medium and psilocybin is excreted; and addressing some of the problems inherent in psilocybin synthesis in prokaryotic systems.

[0117] The present disclosure provides methods of producing psilocybin or a derivative thereof said method comprising a series of enzyme-catalysed reactions wherein each reaction is catalysed by an enzyme expressed by a single strain of microorganism. In some examples, the method comprises three enzyme-catalysed reactions and three respective microorganism strains. In some examples, the method comprises four enzyme-catalysed reactions and four respective microorganism strains. In some examples, the method comprises five enzyme-catalysed reactions and five respective microorganism strains.

[0118] In some examples, the present disclosure provides a method of producing psilocybin or a derivative thereof comprising:(a) preparing a reaction mixture comprising a substrate, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a first enzyme, a second strain of cells or a lysate thereof wherein the second strain is engineered to express a second enzyme, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a third enzyme,(b) incubating the reaction mixture under conditions suitable for the first enzyme to catalyse a reaction converting the substrate to a first intermediate, the second enzyme to catalyse a reaction converting the first intermediate to a second intermediate, and the third enzyme to catalyse a reaction converting the second intermediate to psilocybin or a derivative thereof.

[0119] In some examples, the reaction mixture comprises a fourth enzyme that catalyses a reaction converting one intermediate to another intermediate, or a reaction converting an intermediate to psilocybin or a derivative thereof. The fourth enzyme may be expressed by a fourth strain of cells which is also present in the reaction mixture, or it may be expressed by the first, second or third strain of cells. In some examples, the fourth enzyme is added in an isolated form to the reaction mixture.

[0120] It will be understood that the method may comprise more than three enzyme-catalysed reactions, each catalysed by an enzyme expressed by an engineered strain of cells or a lysate thereof. The methodAU_Active01 27520873v1 CHRISTMmay comprise four, five, six, seven, eight, nine, ten or more enzyme-catalysed reactions. In some examples, the present disclosure provides a method of producing psilocybin or a derivative thereof comprising:(a) preparing a reaction mixture comprising a substrate, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a first enzyme, a second strain of cells or a lysate thereof wherein the second strain is engineered to express a second enzyme, a third strain of cells or a lysate thereof wherein the third strain is engineered to express a third enzyme, and a fourth strain of cells or a lysate thereof wherein the fourth strain is engineered to express a fourth enzyme,(b) incubating the reaction mixture under conditions suitable for the first enzyme to catalyse a reaction converting the substrate to a first intermediate, the second enzyme to catalyse a reaction converting the first intermediate to a second intermediate, the third enzyme to catalyse a reaction converting the second intermediate to a third intermediate, and the fourth enzyme to catalyse a reaction converting the third intermediate to psilocybin or a derivative thereof.

[0121] In some examples, the present disclosure provides a method of producing psilocybin or a derivative thereof comprising:(a) preparing a reaction mixture comprising a substrate, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a first enzyme, a second strain of cells or a lysate thereof wherein the second strain is engineered to express a second enzyme, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a third enzyme,(b) incubating the reaction mixture under conditions suitable for the first enzyme to catalyse a reaction converting the substrate to a first intermediate, the second enzyme to catalyse a reaction converting the first intermediate to a second intermediate, the third enzyme to catalyse a reaction converting the second intermediate to a third intermediate, and a fourth enzyme to catalyse a reaction converting the third intermediate to psilocybin or a derivative thereof. In some examples, the fourth enzyme catalyses a reaction converting one intermediate to another intermediate.AU_Active01 27520873v1 CHRISTM

[0122] Any one or more of the enzymes may be derived from a fungus such as Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, Psilocybe cubensis or Gymnopilus junonius.

[0123] The cells engineered to express a particular enzyme may be prokaryotic cells or eukaryotic cells. The cells may be plant cells, fungal cells, yeast cells, animal cells, algal cells, bacterial cells, archaea cells or protozoan cells. Useful fungi include a fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, S. boulardii, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis, K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, other yeast genera such as Cryptococcus (e.g. C. aerius), Debaromyces (e.g. D. hansenii), Hansenula, Yarrowia (e.g. Y. lipolytica), Zygosaccharomyces (e.g. Z. bailii), Torulaspora (e.g. T. delbrueckii), Schizosaccharomyces (e.g. S. pombe), Brettanomyces (e.g. B. bruxellensis), Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, or Trichoderma. Useful bacteria include bacteria belonging to the genus Bacillus (e.g. B. subtilis), a species belonging to the genus Escherichia (e.g. E. coli), a species belonging to the genus Lactobacillus (e.g. L. casei), a species belonging to the genus Lactococcus (e.g. L. lactis), a species belonging to the genus Corynebacterium (e.g. C. glutamicum), a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas (e.g. P. putida), and a species belonging to the genus Streptomyces (e.g. S. coelicolor). Useful plants include plants belonging to the genus Arabidopsis (e.g. A. thaliana), a species belonging to the genus Zea (e.g. Z. mays), a species belonging to the genus Medicago (e.g. M. truncatula), a species belonging to the genus Nicotiana (e.g. N. tabacum) and a species belonging to the genus Glycine (e.g. G. Max). In some examples, the cells are bacterial cells. In some examples, the cells are E. coli cells.

[0124] In some examples, at least one of the strains carry a defective or suppressed tryptophanase. The tryptophanase may be defective or suppressed due to a genetic mutation (e.g., a substitution, deletion, insertion or inversion) in a gene encoding the tryptophanase. The genetic mutation may be an induced mutation. The tryptophanase may be tryptophanase A or tryptophanase B.

[0125] In some examples, the reaction mixture comprises an additive, such as a membrane permeabilising agent. In some examples the reaction mixture comprises a surfactant. The surfactant may be an ionic surfactant or a non-ionic surfactant. The surfactant may be Tween 20, Tween 80, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton-X, n-Dodecyl p-D-maltoside, digitonin, 3-[(3- cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS) or urea. In some examples, the surfactant is Tween 20. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant. The polar protic solvent may be, for example, acetic acid or an alcohol such as methanol, ethanol, propanol, butanol, methyl-propanol or pentanol. In some examples, the alcohol is / so-propanol. The polar aprotic solvent may be, for example, acetone, N,N- Dimethylformamide (DMF), acetonitrile or dimethyl sulfoxide (DMSO). The surfactant may be ionic or non-ionic. Ionic surfactants may include deoxycholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) or cetyl methyl ammonium bromide (CTAB). Non-ionic detergents may include Triton-X, Tween 20, n-Octyl-p-D-glucopyranoside or Brij-35. In some examples, the membrane permeabilising agent is Tween 20. Those skilled in the art could readily identify a suitable membraneAU_Active01 27520873v1 CHRISTMpermeabilising agent and concentration. In some examples, the membrane permeabilising agent is present in the reaction mixture at a concentration of between about 0.01 % and 5%, such as between about 0.05% and 4%, or between about 0.05% and 3%, or between about 0.1 % and 3%, or between about 0.1 % and 2%, or between about 0.1 % and 1.5%, or between about 0.1 % and 1 %, such as about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1 %.

[0126] In some examples, at least one of the strains of cells is dried or powderised and optionally rehydrated before preparing the reaction mixture. For example, the cells may be spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells are spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol or a combination thereof and preferably magnesium sulfate.

[0127] In some examples, the reaction mixture comprises glycerol at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1.5% or about 2% or about 2.5%. In some examples, the reaction mixture comprises glucose at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1.5% or about 2% or about 2.5%

[0128] In some examples, the reaction mixture comprises NH4CI or a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate. The magnesium salt (eg, MgCh) may be present at a concentration of between about 0.5 mM and 10 mM, or between about 1 mM and 5 mM, or between about 1 mM and 4 mM or between about 1 mM and 3mM, such as about 2 mM. The NH4CI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1 .5 mg / mL, such as about 1 mg / mL. The NH4CI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1 .5 mg / mL, such as about 1 mg / mL. In some examples, the reaction mixture comprises a buffer such as a phosphate buffer. In some examples, the phosphate buffer comprises potassium phosphate or sodium phosphate. The buffer may be present at a concentration of between about 10 mM and 250 mM, such as between about 15 mM and 200 mM, or between about 20 mM and 150 mM, or between about 25 mM and 100 mM, or between about 25 mM and 75 mM, or between about 35 mM and 65 mM, or between about 40 mM and 60 mM, such as about 50 mM.AU_Active01 27520873v1 CHRISTM

[0129] In some examples, the reaction mixture comprises methionine. In some examples, the reaction mixture comprises L-methionine. The methionine may serve to increase endogenous levels of S- adenosylmethionine which may be used as a methyl donor by PsiM.

[0130] In some examples, the pH of the reaction mixture is between about 5 and 9, such as about 5.5, about 6, about 6.5, about 7, about 7.5, about 8 or about 8.5. In some examples, the pH of the reaction mixture is between about 6 and 8.

[0131] In some examples, the reaction mixture is incubated at a temperature of between about 15 °C and 50 °C, such as between about 15 °C and 40 °C, or between about 20 °C and 40 °C, or between about 20 °C and 37 °C, or between about 20 °C and 35 °C, or between about 20 °C and 30 °C, or between about 22.5 °C and 30 °C, or between about 22.5 °C and 27.5 °C. In some examples, the reaction mixture is incubated at a temperature of about 20 °C, or about 21 °C, or about 22 °C, or about 23 °C, or about 24 °C, or about 25 °C, or about 26 °C, or about 27 °C, or about 28 °C, or about 29 °C, or about 30 °C, or about 31 °C, or about 32 °C, or about 33 °C, or about 34 °C, or about 35 °C or about 36 °C, or about 37 °C. In some examples, the reaction mixture is incubated at a temperature of about 25 °C.

[0132] In some examples, the first strain is engineered to express the first enzyme but does not express, or is not engineered to express, the second enzyme or the third enzyme. In some examples, the second strain is engineered to express the second enzyme but does not express, or is not engineered to express, the first enzyme or the third enzyme. In some examples, the third strain is engineered to express the third enzyme but does not express, or is not engineered to express, the first enzyme or the second enzyme.

[0133] In some examples, the first strain is engineered to express a monooxygenase and preferably a reductase but does not express, or is not engineered to express, a kinase or a methyltransferase. In some examples, the second strain is engineered to express a kinase but does not express, or is not engineered to express, a monooxygenase, a reductase or a methyltransferase. In some examples, the third strain is engineered to express a methyltransferase but does not express, or is not engineered to express, a monooxygenase, a reductase or a kinase. The monooxygenase may be a cytochrome P450 monooxygenase (e.g., a Psilocybe cubensis tryptamine 4-hydroxylase cytochrome P450 monooxygenase or a variant or derivative thereof), the reductase may be a cytochrome P450 reductase (e.g., a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof), the kinase may be a Psilocybe cubensis 4-hydroxytryptamine kinase and the methyltransferase may be a N- methyltransferase (e.g., a Psilocybe cubensis methyltransferase).

[0134] In some examples the reaction mixture is substantially free of 4-hydoxyindole and / or serine.A. Production of 4-hvdroxytryptamine and derivatives thereof

[0135] In one aspect, the present disclosure provides a method of producing 4-hydroxytryptamine or a derivative thereof comprising:(a) preparing a reaction mixture comprising tryptamine or a derivative thereof, and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase; andAU_Active01 27520873v1 CHRISTM(b) incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine or derivative thereof to produce 4-hydroxytryptamine or a derivative thereof.

[0136] In some examples, the present disclosure provides a method of producing 4-hydroxytryptamine comprising:(a) preparing a reaction mixture comprising tryptamine, and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase; and(b) incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine.

[0137] The reaction mixture may comprise intact cells. In some examples, the present disclosure provides a method of producing 4-hydroxytryptamine comprising:(a) preparing a reaction mixture comprising tryptamine, and intact cells engineered to express a monooxygenase and a reductase; and(b) incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine.

[0138] The monooxygenase may be a microbial monooxygenase, a prokaryotic monooxygenase, a eukaryotic monooxygenase, an animal monooxygenase, a plant monooxygenase, a bacterial monooxygenase, a protozoan monooxygenase, a fungal monooxygenase, a yeast monooxygenase or an algae monooxygenase.

[0139] In some examples, the monooxygenase is a cytochrome P450 monooxygenase. In some examples, monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.

[0140] In some examples, the monooxygenase is a truncated monooxygenase. In some examples, the monooxygenase comprises an N-terminal truncation. The monooxygenase may comprise an N-terminal truncation of between about 5 amino acids and 40 amino acids, such as between about 10 amino acids and 30 amino acids, or between about 12 amino acids and 25 amino acids, or between about 15 amino acids and 25 amino acids, such as 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids or 24 amino acids. The truncated region may comprise the sequence set forth in SEQ ID NO:6, or a sequence having at least about 70% identity to SEQ ID NO:6, such as a sequence having at least about 75% identity to SEQ ID NO:6, or at least about 80% identity to SEQ ID NO:6, or at least about 85% identity to SEQ ID NO:6, or at least about 90% identity to SEQ ID NO:6, or at least about 91 % identity to SEQ ID NO:6, or at least about 92% identity to SEQ ID NO:6, or at least about 93% identity to SEQ ID NO:6, or at least about 94% identity to SEQ ID NO:6, or at least about 95% identity to SEQ ID NO:6, or at least about 96% identity to SEQ ID NO:6, or at least about 97% identity to SEQ ID NO:6, or at least about 98% identity to SEQ ID NO:6, or at least about 99% identity to SEQ ID NO:6, or 100% identity to SEQ ID NO:6. In some examples, the monooxygenase does not comprise the sequence set forth in SEQ ID NO:6, or a sequence having atAU_Active01 27520873v1 CHRISTMleast about 90% identity to SEQ ID NO:6. In some examples, the truncated region is replaced with a peptide sequence, such as a hydrophilic motif. The peptide sequence may be between about 5 amino acids and 30 amino acids, such as between about 5 amino acids and 20 amino acids, or between about 5 amino acids and 15 amino acids, such as 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 1 1 amino acids, 12 amino acids, 13 amino acids or 14 amino acids. In some examples, the peptide sequence comprises the sequence set forth in SEQ ID NO:7, or a sequence having at least about 70% identity to SEQ ID NO:7, such as a sequence having at least about 75% identity to SEQ ID NO:7, or at least about 80% identity to SEQ ID NO:7, or at least about 85% identity to SEQ ID NO:7, or at least about 90% identity to SEQ ID NOT, or at least about 91 % identity to SEQ ID NOT, or at least about 92% identity to SEQ ID NOT, or at least about 93% identity to SEQ ID NOT, or at least about 94% identity to SEQ ID NOT, or at least about 95% identity to SEQ ID NOT, or at least about 96% identity to SEQ ID NOT, or at least about 97% identity to SEQ ID NOT, or at least about 98% identity to SEQ ID NOT, or at least about 99% identity to SEQ ID NOT, or 100% identity to SEQ ID NOT.

[0141] In some examples, the monooxygenase comprises the sequence set forth in SEQ ID NO:2, or a sequence having at least about 70% identity to SEQ ID NO:2, such as a sequence having at least about 75% identity to SEQ ID NO:2, or at least about 80% identity to SEQ ID NO:2, or at least about 85% identity to SEQ ID NO:2, or at least about 90% identity to SEQ ID NO:2, or at least about 91 % identity to SEQ ID NO:2, or at least about 92% identity to SEQ ID NO:2, or at least about 93% identity to SEQ ID NO:2, or at least about 94% identity to SEQ ID NO:2, or at least about 95% identity to SEQ ID NO:2, or at least about 96% identity to SEQ ID NO:2, or at least about 97% identity to SEQ ID NO:2, or at least about 98% identity to SEQ ID NO:2, or at least about 99% identity to SEQ ID NO:2, or 100% identity to SEQ ID NO:2.

[0142] Enzyme variants can be readily assayed for enzymatic activity using methods well known in the art, and the methods described herein. Variant enzymes may contain deletions, insertions or conservative amino acid substitutions at various locations relative to a parent or reference enzyme. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Those skilled in the art will understand that different amino acids can be grouped based on the properties of their side chains. Such groupings are set out below.

[0143] Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

[0144] Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (eg, histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

[0145] Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (ie, glutamic acid, aspartic acid, arginine, lysine and histidine).AU_Active01 27520873v1 CHRISTM

[0146] Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

[0147] Neutral / polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral / polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

[0148] Certain amino acids may also be characterized as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, “small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the a-amino group, as well as the a-carbon. For the purposes of the present disclosure, however, proline is considered to be a “small” amino acid.

[0149] Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, subclassification according to this scheme is presented in Table 1 .AU_Active01 27520873v1 CHRISTMTable 1. Amino acid sub-groupings

[0150] Conservative amino acid substitutions are also grouped based on amino acid side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change alters the activity of an enzyme can readily be determined using known assays. Conservative substitutions are shown in Table 2 under the heading of exemplary and preferred substitutions.Table 2. Exemplary amino acid substitutionsAU_Active01 27520873v1 CHRISTM

[0151] Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G. Biochemistry, third edition, Wm.C. Brown Publishers (1993).

[0152] In some examples, the cells engineered to express a monooxygenase are not engineered to express a kinase and / or a methyltransferase.

[0153] In some examples, the cells are engineered to express a monooxygenase and a reductase. The reductase may be a microbial reductase, a prokaryotic reductase, a eukaryotic reductase, an animal reductase, a plant reductase, a bacterial reductase, a protozoan reductase, a fungal reductase, a yeast reductase or an algae reductase.

[0154] Expression of the monooxygenase and / or the reductase may be induced. For example, the cells engineered to express the monooxygenase and / or the reductase may be cultured in autoinduction medium. Expression or activity of the monooxygenase and / or the reductase may be induced by aminolevulinic acid. Expression of the monooxygenase and / or the reductase may be induced by an enzymatic digest of soy (soytone). In some examples, expression is induced by isopropyl p-D-1- thiogalactopyranoside (IPTG), methionine, lactose or arabinose.

[0155] The reductase may transfer electrons from NADH / NADPH to the monooxygenase. In some examples, the reductase is a cytochrome P450 reductase. In some examples, the reductase is aAU_Active01 27520873v1 CHRISTMPsilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof. In some examples, the reductase transfers electrons from NADH / NADPH to the monooxygenase.

[0156] In some examples, the reductase comprises the sequence set forth in SEQ ID NO:3, or a sequence having at least about 70% identity to SEQ ID NO:3, such as a sequence having at least about 75% identity to SEQ ID NO:3, or at least about 80% identity to SEQ ID NO:3, or at least about 85% identity to SEQ ID NO:3, or at least about 90% identity to SEQ ID NO:3, or at least about 91% identity to SEQ ID NO:3, or at least about 92% identity to SEQ ID NO:3, or at least about 93% identity to SEQ ID NO:3, or at least about 94% identity to SEQ ID NO:3, or at least about 95% identity to SEQ ID NO:3, or at least about 96% identity to SEQ ID NO:3, or at least about 97% identity to SEQ ID NO:3, or at least about 98% identity to SEQ ID NO:3, or at least about 99% identity to SEQ ID NO:3, or 100% identity to SEQ ID NO:3.

[0157] The monooxygenase and reductase may be expressed from separate genes engineered into each cell. In some examples, the monooxygenase and reductase are expressed as a fusion protein, optionally comprising a cleavage site between the monooxygenase and the reductase. In some examples, the monooxygenase and reductase are encoded in a single operon. The operon may comprise separate ribosome binding sites that initiate translation of the monooxygenase and the reductase. In some examples, the monooxygenase and the reductase are encoded by one or more nucleic acids codon optimised for expression in the cells.

[0158] The cells engineered to express a monooxygenase and a reductase may be prokaryotic cells or eukaryotic cells. The cells may be plant cells, fungal cells, yeast cells, animal cells, algal cells, bacterial cells, archaea cells or protozoan cells. Useful fungi include a fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, S. boulardii, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis, K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, other yeast genera such as Cryptococcus (e.g. C. aerius), Debaromyces (e.g. D. hansenii), Hansenula, Yarrowia (e.g. Y. lipolytica), Zygosaccharomyces (e.g. Z. bailii), Torulaspora (e.g. T. delbrueckii), Schizosaccharomyces (e.g. S. pombe), Brettanomyces (e.g. B. bruxellensis), Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, or Trichoderma. Useful bacteria include bacteria belonging to the genus Bacillus (e.g. B. subtilis), a species belonging to the genus Escherichia (e.g. E. coli), a species belonging to the genus Lactobacillus (e.g. L. casei), a species belonging to the genus Lactococcus (e.g. L. lactis), a species belonging to the genus Corynebacterium (e.g. C. glutamicum), a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas (e.g. P. putida), and a species belonging to the genus Streptomyces (e.g. S. coelicolor). Useful plants include plants belonging to the genus Arabidopsis (e.g. A. thaliana), a species belonging to the genus Zea (e.g. Z. mays), a species belonging to the genus Medicago (e.g. M. truncatula), a species belonging to the genus Nicotiana (e.g. N. tabacum) and a species belonging to the genus Glycine (e.g. G. Max). In some examples, the cells are bacterial cells. In some examples, the cells are E. coli cells.AU_Active01 27520873v1 CHRISTM

[0159] In some examples, the cells carry a defective or suppressed tryptophanase. The tryptophanase may be defective or suppressed due to a genetic mutation (e.g., a substitution, deletion, insertion or inversion) in a gene encoding the tryptophanase. The genetic mutation may be an induced mutation. The tryptophanase may be tryptophanase A or tryptophanase B.

[0160] In some examples, the reaction mixture comprises an additive, such as a membrane permeabilising agent. In some examples the reaction mixture comprises a surfactant. The surfactant may be an ionic surfactant or a non-ionic surfactant. The surfactant may be Tween 20, Tween 80, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton-X, n-Dodecyl p-D-maltoside, digitonin, 3-[(3- cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS) or urea. In some examples, the surfactant is Tween 20. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant. The polar protic solvent may be, for example, acetic acid or an alcohol such as methanol, ethanol, propanol, butanol, methyl-propanol or pentanol. In some examples, the alcohol is / so-propanol. The polar aprotic solvent may be, for example, acetone, / V, / V- Dimethylformamide (DMF), acetonitrile or dimethyl sulfoxide (DMSO). The surfactant may be ionic or non-ionic. Ionic surfactants may include deoxycholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) or cetyl methyl ammonium bromide (CTAB). Non-ionic detergents may include Triton-X, Tween 20, n-Octyl-p-D-glucopyranoside or Brij-35. In some examples, the membrane permeabilising agent is Tween 20. Those skilled in the art could readily identify a suitable membrane permeabilising agent and concentration. In some examples, the membrane permeabilising agent is present in the reaction mixture at a concentration of between about 0.01 % and 5%, such as between about 0.05% and 4%, or between about 0.05% and 3%, or between about 0.1 % and 3%, or between about 0.1 % and 2%, or between about 0.1 % and 1.5%, or between about 0.1 % and 1 %, such as about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1 %.

[0161] In some examples, the cells are dried or powderised and optionally rehydrated before preparing the reaction mixture. For example, the cells may be spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells are spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol or a combination thereof and preferably magnesium sulfate.

[0162] In some examples, the reaction mixture comprises glycerol at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1 .5% or about 2% or about 2.5%. In some examples, the reaction mixture comprises glucose at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1 .5% or about 2% or about 2.5%. In some examples, the reaction mixture comprises aminolevulinic acid.AU_Active01 27520873v1 CHRISTM

[0163] In some examples, the reaction mixture comprises a salt such NH4CI or a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate. The magnesium salt (eg, MgCh) may be present at a concentration of between about 0.5 mM and 10 mM, or between about 1 mM and 5 mM, or between about 1 mM and 4 mM or between about 1 mM and 3mM, such as about 2 mM. The NH4CI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1 .5 mg / mL, such as about 1 mg / mL. The NH4CI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1 .5 mg / mL, such as about 1 mg / mL. In some examples, the reaction mixture comprises a buffer such as a phosphate buffer. In some examples, the phosphate buffer comprises potassium phosphate or sodium phosphate. The phosphate buffer may be present at a concentration of between about 10 mM and 250 mM, such as between about 15 mM and 200 mM, or between about 20 mM and 150 mM, or between about 25 mM and 100 mM, or between about 25 mM and 75 mM, or between about 35 mM and 65 mM, or between about 40 mM and 60 mM, such as about 50 mM.

[0164] In some examples, the pH of the reaction mixture is between about 5 and 9, such as about 5.5, about 6, about 6.5, about 7, about 7.5, about 8 or about 8.5. In some examples, the pH of the reaction mixture is between about 6 and 8.

[0165] In some examples, the reaction mixture is incubated at a temperature of between about 15 °C and 50 °C, such as between about 15 °C and 40 °C, or between about 20 °C and 40 °C, or between about 20 °C and 37 °C, or between about 20 °C and 35 °C, or between about 20 °C and 30 °C, or between about 22.5 °C and 30 °C, or between about 22.5 °C and 27.5 °C. In some examples, the reaction mixture is incubated at a temperature of about 20 °C, or about 21 °C, or about 22 °C, or about 23 °C, or about 24 °C, or about 25 °C, or about 26 °C, or about 27 °C, or about 28 °C, or about 29 °C, or about 30 °C, or about 31 °C, or about 32 °C, or about 33 °C, or about 34 °C, or about 35 °C or about 36 °C, or about 37 °C. In some examples, the reaction mixture is incubated at a temperature of about 25 °C.

[0166] In some examples, the reaction mixture is substantially free of 4-hydoxyindole and / or serine.

[0167] In some examples, the method further comprises producing norbaeocystin as described in Section B below. That is, the present disclosure provides methods of producing norbaeocystin that combine the methods described in Section A and the methods described in Section B.B. Production of norbaeocystin and derivatives thereof

[0168] In another aspect, the present disclosure provides a method of producing norbaeocystin or a derivative thereof comprising:(a) preparing a reaction mixture comprising4-hydroxytryptamine or a derivative thereof, and cells or a lysate thereof wherein the cells are engineered to express a kinase; and(b) incubating the reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof.AU_Active01 27520873v1 CHRISTM

[0169] In some examples, the present disclosure provides a method of producing norbaeocystin comprising:(a) preparing a reaction mixture comprising4-hydroxytryptamine, and cells or a lysate thereof wherein the cells are engineered to express a kinase; and(b) incubating the reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.

[0170] The reaction mixture may comprise intact cells. In some examples, the present disclosure provides a method of producing norbaeocystin comprising:(a) preparing a reaction mixture comprising4-hydroxytryptamine, and intact cells engineered to express a kinase; and(b) incubating the reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.

[0171] The kinase may be a microbial kinase, a prokaryotic kinase, a eukaryotic kinase, an animal kinase, a plant kinase, a bacterial kinase, a protozoan kinase, a fungal kinase, a yeast kinase or an algae kinase.

[0172] The kinase is preferably a 4-hydroxytryptamine kinase. In some examples, the kinase is a Psilocybe cubensis 4-hydroxytryptamine kinase or a variant or derivative thereof.

[0173] In some examples, the kinase comprises the sequence set forth in SEQ ID NO:4, or a sequence having at least about 70% identity to SEQ ID NO:4, such as a sequence having at least about 75% identity to SEQ ID NO:4, or at least about 80% identity to SEQ ID NO:4, or at least about 85% identity to SEQ ID NO:4, or at least about 90% identity to SEQ ID NO:4, or at least about 91 % identity to SEQ ID NO:4, or at least about 92% identity to SEQ ID NO:4, or at least about 93% identity to SEQ ID NO:4, or at least about 94% identity to SEQ ID NO:4, or at least about 95% identity to SEQ ID NO:4, or at least about 96% identity to SEQ ID NO:4, or at least about 97% identity to SEQ ID NO:4, or at least about 98% identity to SEQ ID NO:4, or at least about 99% identity to SEQ ID NO:4, or 100% identity to SEQ ID NO:4.

[0174] Expression of the kinase may be induced. For example, the cells engineered to express the kinase may be cultured in autoinduction medium. Expression of the kinase may be induced by methionine. Expression of the kinase may be induced by an enzymatic digest of soy (soytone). In some examples, expression is induced by isopropyl p-D-1 -thiogalactopyranoside (IPTG), aminolevulinic acid, lactose or arabinose.

[0175] In some examples, the reaction mixture further comprises adenosine triphosphate (ATP) which provides a phosphate for the phosphorylation. In some examples, the kinase is an ATP-dependent 4- hydroxytryptamine kinase that catalyses 4-O-phosphorylation of the 4-hydroxytryptamine or a derivative thereof to produce norbaeocystin or a derivative thereof.

[0176] In some examples, the kinase is encoded by a nucleic acid codon optimised for expression in the cells.AU_Active01 27520873v1 CHRISTM

[0177] In some examples, the cells engineered to express a kinase are not engineered to express a monooxygenase, a reductase or a methyltransferase.

[0178] The cells engineered to express a kinase may be prokaryotic cells or eukaryotic cells. The cells may be plant cells, fungal cells, yeast cells, animal cells, algal cells, bacterial cells, archaea cells or protozoan cells. In some examples, the cells are bacterial cells. Useful fungi include a fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, S. boulardii, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis, K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, other yeast genera such as Cryptococcus (e.g. C. aerius), Debaromyces (e.g. D. hansenii), Hansenula, Yarrowia (e.g. Y. lipolytica), Zygosaccharomyces (e.g. Z. bailii), Torulaspora (e.g. T. delbrueckii), Schizosaccharomyces (e.g. S. pombe), Brettanomyces (e.g. B. bruxellensis), Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, or Trichoderma. Useful bacteria include bacteria belonging to the genus Bacillus (e.g. B. subtilis), a species belonging to the genus Escherichia (e.g. E. coli), a species belonging to the genus Lactobacillus (e.g. L. casei), a species belonging to the genus Lactococcus (e.g. L. lactis), a species belonging to the genus Corynebacterium (e.g. C. glutamicum), a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas (e.g. P. putida), and a species belonging to the genus Streptomyces (e.g. S. coelicolor). Useful plants include plants belonging to the genus Arabidopsis (e.g. A. thaliana), a species belonging to the genus Zea (e.g. Z. mays), a species belonging to the genus Medicago (e.g. M. truncatula), a species belonging to the genus Nicotiana (e.g. N. tabacum) and a species belonging to the genus Glycine (e.g. G. Max). In some examples, the cells are bacterial cells. In some examples, the cells are E. coli cells.

[0179] In some examples, the cells carry a defective or suppressed tryptophanase. The tryptophanase may be defective or suppressed due to a genetic mutation (e.g., a substitution, deletion, insertion or inversion) in a gene encoding the tryptophanase. The genetic mutation may be an induced mutation. The tryptophanase may be tryptophanase A or tryptophanase B.

[0180] In some examples, the reaction mixture comprises an additive, such as a membrane permeabilising agent. In some examples the reaction mixture comprises a surfactant. The surfactant may be an ionic surfactant or a non-ionic surfactant. The surfactant may be Tween 20, Tween 80, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton-X, n-Dodecyl p-D-maltoside, digitonin, 3-[(3- cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS) or urea. In some examples, the surfactant is Tween 20. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant. The polar protic solvent may be, for example, acetic acid or an alcohol such as methanol, ethanol, propanol, butanol, methyl-propanol or pentanol. In some examples, the alcohol is / so-propanol. The polar aprotic solvent may be, for example, acetone, N,N- Dimethylformamide (DMF), acetonitrile or dimethyl sulfoxide (DMSO). The surfactant may be ionic or non-ionic. Ionic surfactants may include deoxycholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) or cetyl methyl ammonium bromide (CTAB). Non-ionic detergents may include Triton-X, Tween 20, n-Octyl-p-D-glucopyranoside or Brij-35. In some examples, the membraneAU_Active01 27520873v1 CHRISTMpermeabilising agent is Tween 20. Those skilled in the art could readily identify a suitable membrane permeabilising agent and concentration. In some examples, the membrane permeabilising agent is present in the reaction mixture at a concentration of between about 0.01% and 5%, such as between about 0.05% and 4%, or between about 0.05% and 3%, or between about 0.1 % and 3%, or between about 0.1 % and 2%, or between about 0.1 % and 1.5%, or between about 0.1 % and 1 %, such as about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1 %.

[0181] In some examples, the cells are dried or powderised and optionally rehydrated before preparing the reaction mixture. For example, the cells may be spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells are spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol or a combination thereof and preferably magnesium sulfate.

[0182] In some examples, the reaction mixture comprises glycerol at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1.5% or about 2% or about 2.5%. In some examples, the reaction mixture comprises glucose at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1.5% or about 2% or about 2.5%.

[0183] In some examples, the reaction mixture further comprises adenosine triphosphate.

[0184] In some examples, the reaction mixture comprises a salt such as NFUCI or a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate. The magnesium salt (eg, MgCh) may be present at a concentration of between about 0.5 mM and 10 mM, or between about 1 mM and 5 mM, or between about 1 mM and 4 mM or between about 1 mM and 3mM, such as about 2 mM. The NH4CI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1 .5 mg / mL, such as about 1 mg / mL. In some examples, the reaction mixture comprises a buffer such as a phosphate buffer. In some examples, the phosphate buffer comprises potassium phosphate or sodium phosphate. The phosphate buffer may be present at a concentration of between about 10 mM and 250 mM, such as between about 15 mM and 200 mM, or between about 20 mM and 150 mM, or between about 25 mM and 100 mM, or between about 25 mM and 75 mM, or between about 35 mM and 65 mM, or between about 40 mM and 60 mM, such as about 50 mM.

[0185] In some examples, the pH of the reaction mixture is between about 5 and 9, such as about 5.5, about 6, about 6.5, about 7, about 7.5, about 8 or about 8.5. In some examples, the pH of the reaction mixture is between about 6 and 8.AU_Active01 27520873v1 CHRISTM

[0186] In some examples, the reaction mixture is incubated at a temperature of between about 15 °C and 50 °C, such as between about 15 °C and 40 °C, or between about 20 °C and 40 °C, or between about 20 °C and 37 °C, or between about 20 °C and 35 °C, or between about 20 °C and 30 °C, or between about 22.5 °C and 30 °C, or between about 22.5 °C and 27.5 °C. In some examples, the reaction mixture is incubated at a temperature of about 20 °C, or about 21 °C, or about 22 °C, or about 23 °C, or about 24 °C, or about 25 °C, or about 26 °C, or about 27 °C, or about 28 °C, or about 29 °C, or about 30 °C, or about 31 °C, or about 32 °C, or about 33 °C, or about 34 °C, or about 35 °C or about 36 °C, or about 37 °C. In some examples, the reaction mixture is incubated at a temperature of about 25 °C.

[0187] In some examples, the reaction mixture further comprises tryptamine or a derivative thereof, and cells engineered to express a monooxygenase and optionally a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine or derivative thereof to produce the 4-hydroxytryptamine or a derivative thereof; and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof.

[0188] In some examples, the reaction mixture is substantially free of 4-hydoxyindole and / or serine.

[0189] The 4-hydroxytryptamine may be produced according to any of the methods described in Section A above. That is, the methods described in Section A may be combined with the methods described in Section B to produce norbaeocystin.

[0190] In some examples, the present disclosure provides a method of producing norbaeocystin or a derivative thereof comprising:(a) preparing a reaction mixture comprising tryptamine or a derivative thereof, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and optionally a reductase, and a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase; and(b) incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine or derivative thereof to produce 4-hydroxytryptamine or a derivative thereof, and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof.

[0191] A strain is a subtype of a species and may include a natural or induced genetic variant, or a genetically modified organism. The term strain is commonly used in reference to microorganisms and viruses, but can also refer to a plant (where the term may be synonymous with cultivar or variety) or an animal. Two different strains may be derived from a single strain, for example, by genetic modification of a parental strain. In such examples, one strain may be distinguished from another strain by the presence or absence of a gene encoding an enzyme capable of catalysing a reaction in a psilocybin biosynthesis pathway. The parental strain may be chosen because it possesses advantageous characteristics such as a high growth rate, stress tolerance etc.AU_Active01 27520873v1 CHRISTMC. Production of psilocybin and derivatives thereof

[0192] In one aspect, the present disclosure provides a method of producing psilocybin or a derivative thereof comprising:(a) preparing a reaction mixture comprising norbaeocystin or a derivative thereof, and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof, and the methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.

[0193] In some examples, the present disclosure provides a method of producing psilocybin comprising:(a) preparing a reaction mixture comprising norbaeocystin, and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

[0194] The reaction mixture may comprise intact cells. In some examples, the present disclosure provides a method of producing psilocybin comprising:(a) preparing a reaction mixture comprising norbaeocystin, and intact cells engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

[0195] The methyltransferase may be a microbial methyltransferase, a prokaryotic methyltransferase, a eukaryotic methyltransferase, an animal methyltransferase, a plant methyltransferase, a bacterial methyltransferase, a protozoan methyltransferase, a fungal methyltransferase, a yeast methyltransferase or an algae methyltransferase.

[0196] The methyltransferase may be a / V-methyltransferase. In some examples, the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.

[0197] In some examples, the methyltransferase comprises the sequence set forth in SEQ ID NO:5, or a sequence having at least about 70% identity to SEQ ID NO:5, such as a sequence having at least about 75% identity to SEQ ID NO:5, or at least about 80% identity to SEQ ID NO:5, or at least about 85% identity to SEQ ID NO:5, or at least about 90% identity to SEQ ID NO:5, or at least about 91 % identity toAU_Active01 27520873v1 CHRISTMSEQ ID NO:5, or at least about 92% identity to SEQ ID NO:5, or at least about 93% identity to SEQ ID NO:5, or at least about 94% identity to SEQ ID NO:5, or at least about 95% identity to SEQ ID NO:5, or at least about 96% identity to SEQ ID NO:5, or at least about 97% identity to SEQ ID NO:5, or at least about 98% identity to SEQ ID NO:5, or at least about 99% identity to SEQ ID NO:5, or 100% identity to SEQ ID NO:5.

[0198] Expression of the methyltransferase may be induced. For example, the cells engineered to express the methyltransferase may be cultured in autoinduction medium. Expression of the methyltransferase may be induced by methionine. Expression of the methyltransferase may be induced by an enzymatic digest of soy (soytone). In some examples, expression is induced by isopropyl p-D-1- thiogalactopyranoside (IPTG), aminolevulinic acid, lactose or arabinose.

[0199] In some examples, the reaction mixture further comprises S-adenosylmethionine which provides a methyl for the methylation reactions. In some examples, the reaction mixture comprises adenosine triphosphate and L-methionine which facilitates the generation of S-adenosylmethionine in the reaction mixture. It will be understood that S-adenosylmethionine need not be added directly to the reaction mixture; rather, comprises adenosine triphosphate and L-methionine may be directly added and may, in turn, give rise to S-adenosylmethionine. In some examples, the reaction mixture comprises methionine. The methionine may serve to increase endogenous levels of S-adenosylmethionine which may be used as a methyl donor by PsiM.

[0200] In some examples, the methyltransferase is encoded by a nucleic acid codon optimised for expression in the cells.

[0201] In some examples, the cells engineered to express a methyltransferase are not engineered to express a monooxygenase, a reductase or a kinase.

[0202] The cells engineered to express a methyltransferase may be prokaryotic cells or eukaryotic cells. The cells may be plant cells, fungal cells, yeast cells, animal cells, algal cells, bacterial cells, archaea cells or protozoan cells. In some examples, the cells are bacterial cells. Useful fungi include a fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, S. boulardii, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis, K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, other yeast genera such as Cryptococcus (e.g. C. aerius), Debaromyces (e.g. D. hansenii), Hansenula, Yarrowia (e.g. Y. lipolytica), Zygosaccharomyces (e.g. Z. bailii), Torulaspora (e.g. T. delbrueckii), Schizosaccharomyces (e.g. S. pombe), Brettanomyces (e.g. B. bruxellensis), Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, or Trichoderma. Useful bacteria include bacteria belonging to the genus Bacillus (e.g. B. subtilis), a species belonging to the genus Escherichia (e.g. E. coli), a species belonging to the genus Lactobacillus (e.g. L. casei), a species belonging to the genus Lactococcus (e.g. L. lactis), a species belonging to the genus Corynebacterium (e.g. C. glutamicum), a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas (e.g. P. putida), and a species belonging to the genus Streptomyces (e.g. S. coelicolor). Useful plants include plants belonging to the genusAU_Active01 27520873v1 CHRISTMArabidopsis (e.g. A. thaliana), a species belonging to the genus Zea (e.g. Z. mays), a species belonging to the genus Medicago (e.g. M. truncatula), a species belonging to the genus Nicotiana (e.g. N. tabacum) and a species belonging to the genus Glycine (e.g. G. Max). In some examples, the cells are bacterial cells. In some examples, the cells are E. coli cells.

[0203] In some examples, the cells carry a defective or suppressed tryptophanase. The tryptophanase may be defective or suppressed due to a genetic mutation (e.g., a substitution, deletion, insertion or inversion) in a gene encoding the tryptophanase. The genetic mutation may be an induced mutation. The tryptophanase may be tryptophanase A or tryptophanase B.

[0204] In some examples, the reaction mixture comprises an additive, such as a membrane permeabilising agent. In some examples the reaction mixture comprises a surfactant. The surfactant may be an ionic surfactant or a non-ionic surfactant. The surfactant may be Tween 20, Tween 80, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton-X, n-Dodecyl p-D-maltoside, digitonin, 3-[(3- cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS) or urea. In some examples, the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant. The polar protic solvent may be, for example, acetic acid or an alcohol such as methanol, ethanol, propanol, butanol, methyl-propanol or pentanol. In some examples, the alcohol is / so-propanol. The polar aprotic solvent may be, for example, acetone, / V, / V-Dimethylformamide (DMF), acetonitrile or dimethyl sulfoxide (DMSO). The surfactant may be ionic or non-ionic. Ionic surfactants may include deoxycholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) or cetyl methyl ammonium bromide (CTAB). Non-ionic detergents may include Triton-X, Tween 20, n-Octyl-p-D-glucopyranoside or Brij-35. In some examples, the membrane permeabilising agent is Tween 20. Those skilled in the art could readily identify a suitable membrane permeabilising agent and concentration. In some examples, the membrane permeabilising agent is present in the reaction mixture at a concentration of between about 0.01 % and 5%, such as between about 0.05% and 4%, or between about 0.05% and 3%, or between about 0.1 % and 3%, or between about 0.1 % and 2%, or between about 0.1 % and 1.5%, or between about 0.1 % and 1 %, such as about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1 %.

[0205] In some examples, the cells are dried or powderised and optionally rehydrated before preparing the reaction mixture. For example, the cells may be spray dried, freeze dried, air dried or vacuum dried. In some examples, dried cells are mixed with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol or a combination thereof and preferably magnesium sulfate.

[0206] In some examples, the reaction mixture comprises glycerol at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1 .5% or about 2% or about 2.5%. In some examples, the reaction mixture comprises glucose at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5%AU_Active01 27520873v1 CHRISTMand 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1.5% or about 2% or about 2.5%.

[0207] In some examples, the reaction mixture comprises a salt such as NH4CI or a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate. The magnesium salt (eg, MgCh) may be present at a concentration of between about 0.5 mM and 10 mM, or between about 1 mM and 5 mM, or between about 1 mM and 4 mM or between about 1 mM and 3mM, such as about 2 mM. The NH4CI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1 .5 mg / mL, such as about 1 mg / mL. In some examples, the reaction mixture comprises a buffer such as a phosphate buffer. In some examples, the phosphate buffer comprises potassium phosphate or sodium phosphate. The buffer may be present at a concentration of between about 10 mM and 250 mM, such as between about 15 mM and 200 mM, or between about 20 mM and 150 mM, or between about 25 mM and 100 mM, or between about 25 mM and 75 mM, or between about 35 mM and 65 mM, or between about 40 mM and 60 mM, such as about 50 mM.

[0208] In some examples, the pH of the reaction mixture is between about 5 and 9, such as about 5.5, about 6, about 6.5, about 7, about 7.5, about 8 or about 8.5. In some examples, the pH of the reaction mixture is between about 6 and 8.

[0209] In some examples, the reaction mixture is incubated at a temperature of between about 15 °C and 50 °C, such as between about 15 °C and 40 °C, or between about 20 °C and 40 °C, or between about 20 °C and 37 °C, or between about 20 °C and 35 °C, or between about 20 °C and 30 °C, or between about 22.5 °C and 30 °C, or between about 22.5 °C and 27.5 °C. In some examples, the reaction mixture is incubated at a temperature of about 20 °C, or about 21 °C, or about 22 °C, or about 23 °C, or about 24 °C, or about 25 °C, or about 26 °C, or about 27 °C, or about 28 °C, or about 29 °C, or about 30 °C, or about 31 °C, or about 32 °C, or about 33 °C, or about 34 °C, or about 35 °C or about 36 °C, or about 37 °C. In some examples, the reaction mixture is incubated at a temperature of about 25 °C.

[0210] In some examples, the reaction mixture further comprises 4-hydroxytryptamine or a derivative thereof, and cells engineered to express a kinase or a lysate thereof, wherein the reaction mixture is incubated under conditions suitable for: the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce the norbaeocystin or a derivative thereof; the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof; and the methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.

[0211] The norbaeocystin may be produced according to any of the methods described in Section B above. That is, the methods described in Section B may be performed in combination with the methods described in Section C.

[0212] In some examples, the present disclosure provides a method of producing psilocybin or a derivative thereof comprising:AU_Active01 27520873v1 CHRISTM(a) preparing a reaction mixture comprising4-hydroxytryptamine or a derivative thereof, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a kinase, and a second strain cells or a lysate thereof wherein the second strain is engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof, the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof; and the methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.

[0213] In some examples, the reaction mixture further comprises tryptamine or a derivative thereof, and cells engineered to express a monooxygenase and optionally a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine or derivative thereof to produce the 4-hydroxytryptamine or a derivative thereof; the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof; the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof; and the methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.

[0214] In some examples, the reaction mixture is substantially free of 4-hydoxyindole and / or serine.

[0215] The method may further comprise production of 4-hydroxytryptamine according to any of the methods described in Section A above. That is, the methods described in Section A and Section B may be performed in combination with the methods described in Section C.

[0216] In some examples, the present disclosure provides a method of producing psilocybin or a derivative thereof comprising:(a) preparing a reaction mixture comprising tryptamine or a derivative thereof, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase, a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable forAU_Active01 27520873v1 CHRISTMthe monooxygenase to catalyse hydroxylation of the tryptamine or derivative thereof to produce 4-hydroxytryptamine or a derivative thereof, the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof, the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof, and the methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.D. Two- and three-pot conversion

[0217] By expressing different enzymes in different bacteria, the rate of the psilocybin biosynthesis pathway can be controlled by using different amounts of each engineered bacterial strain. For example, if one step is limiting the rate of the overall reaction pathway, then the reaction mixture may include more biomass from the bacterial strain engineered to express the enzyme that catalyses that step, relative to strains engineered to express other enzymes in the pathway. Each strain of bacterium may be mixed together in a single reaction mixture. The strains may be added simultaneously or sequentially. In some examples, different steps of the psilocybin biosynthesis pathway are performed across separate reaction mixtures, each mixture comprising one or more strains of bacterium. In examples where different steps of the psilocybin biosynthesis pathway are performed across different reaction mixtures, the product of one reaction may be purified and added to a subsequent reaction mixture where it becomes a substrate.

[0218] The present disclosure provides one-pot and two-pot methods for converting tryptamine or a derivative thereof to psilocybin or a derivative thereof. A two-pot method for producing psilocybin or a derivative thereof may comprise:(a) preparing a first reaction mixture comprising tryptamine or a derivative thereof, and a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase,(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine or derivative thereof to produce 4-hydroxytryptamine or a derivative thereof;(c) preparing a second reaction mixture by combining the 4-hydroxytryptamine or derivative thereof with a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof, the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof, andAU_Active01 27520873v1 CHRISTMthe methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.

[0219] In some examples, the 4-hydroxytryptamine or a derivative thereof is purified from the first reaction mixture before combining with the second strain and third strain of cells.

[0220] In some examples, the present disclosure provides a two-pot method for producing psilocybin or a derivative thereof comprising:(a) preparing a first reaction mixture comprising tryptamine or a derivative thereof, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase, and a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase,(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine or derivative thereof to produce 4-hydroxytryptamine or a derivative thereof, and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine or derivative thereof to produce norbaeocystin or a derivative thereof,(c) preparing a second reaction mixture by combining the norbaeocystin or derivative thereof with a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof, and the methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.

[0221] In some examples, the norbaeocystin or a derivative thereof is purified before combining with the third strain of cells.

[0222] In some examples, the present disclosure provides a three-pot method for producing psilocybin or a derivative thereof comprising:(a) preparing a first reaction mixture comprising tryptamine or a derivative thereof, and a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase;(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine;(c) preparing a second reaction mixture by combining the 4-hydroxytryptamine with a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase;(d) incubating the second reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin;(e) preparing a third reaction mixture by combining the norbaeocystin with a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase; andAU_Active01 27520873v1 CHRISTM(f) incubating the third reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin or derivative thereof to produce baeocystin or a derivative thereof, and the methyltransferase to catalyse methylation of the baeocystin or derivative thereof to produce psilocybin or a derivative thereof.

[0223] In some examples, the 4-hydroxytryptamine is purified before combining with the second strain of cells. In some examples, the norbaeocystin is purified before combining with the third strain of cells.

[0224] The monooxygenase, the reductase, the kinase and the methyltransferase may be a monooxygenase, reductase, kinase or methyltransferase as described above.

[0225] The cells engineered to express a particular enzyme may be prokaryotic cells or eukaryotic cells. The cells may be plant cells, fungal cells, yeast cells, animal cells, algal cells, bacterial cells, archaea cells or protozoan cells. Useful fungi include a fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, S. boulardii, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis, K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, other yeast genera such as Cryptococcus (e.g. C. aerius), Debaromyces (e.g. D. hansenii), Hansenula, Yarrowia (e.g. Y. lipolytica), Zygosaccharomyces (e.g. Z. bailii), Torulaspora (e.g. T. delbrueckii), Schizosaccharomyces (e.g. S. pombe), Brettanomyces (e.g. B. bruxellensis), Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, or Trichoderma. Useful bacteria include bacteria belonging to the genus Bacillus (e.g. B. subtilis), a species belonging to the genus Escherichia (e.g. E. coli), a species belonging to the genus Lactobacillus (e.g. L. casei), a species belonging to the genus Lactococcus (e.g. L. lactis), a species belonging to the genus Corynebacterium (e.g. C. glutamicum), a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas (e.g. P. putida), and a species belonging to the genus Streptomyces (e.g. S. coelicolor). Useful plants include plants belonging to the genus Arabidopsis (e.g. A. thaliana), a species belonging to the genus Zea (e.g. Z. mays), a species belonging to the genus Medicago (e.g. M. truncatula), a species belonging to the genus Nicotiana (e.g. N. tabacum) and a species belonging to the genus Glycine (e.g. G. Max). In some examples, the cells are bacterial cells. In some examples, the cells are E. coli cells.

[0226] In some examples, at least one of the strains carry a defective or suppressed tryptophanase. The tryptophanase may be defective or suppressed due to a genetic mutation (e.g., a substitution, deletion, insertion or inversion) in a gene encoding the tryptophanase. The genetic mutation may be an induced mutation. The tryptophanase may be tryptophanase A or tryptophanase B.

[0227] In some examples, at least one of the reaction mixtures comprises an additive, such as a membrane permeabilising agent. In some examples the reaction mixture comprises a surfactant. The surfactant may be an ionic surfactant or a non-ionic surfactant. The surfactant may be Tween 20, Tween 80, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton-X, n-Dodecyl p-D-maltoside, digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or urea. In some examples, the surfactant is Tween 20. In some examples, the membrane permeabilising agent is anAU_Active01 27520873v1 CHRISTMalcohol, a polar protic solvent, a polar aprotic solvent or a surfactant. The polar protic solvent may be, for example, acetic acid or an alcohol such as methanol, ethanol, propanol, butanol, methyl-propanol or pentanol. In some examples, the alcohol is / so-propanol. The polar aprotic solvent may be, for example, acetone, / V, / V-Dimethylformamide (DMF), acetonitrile or dimethyl sulfoxide (DMSO). The surfactant may be ionic or non-ionic. Ionic surfactants may include deoxycholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) or cetyl methyl ammonium bromide (CTAB). Non-ionic detergents may include Triton-X, Tween 20, n-Octyl-p-D-glucopyranoside or Brij-35. In some examples, the membrane permeabilising agent is Tween 20. Those skilled in the art could readily identify a suitable membrane permeabilising agent and concentration. In some examples, the membrane permeabilising agent is present in the reaction mixture at a concentration of between about 0.01 % and 5%, such as between about 0.05% and 4%, or between about 0.05% and 3%, or between about 0.1 % and 3%, or between about 0.1 % and 2%, or between about 0.1% and 1.5%, or between about 0.1 % and 1 %, such as about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1 %.

[0228] In some examples, at least one of the strains of cells is dried or powderised and optionally rehydrated before preparing the reaction mixture. For example, the cells may be spray dried, freeze dried, air dried or vacuum dried. In some examples, the cells are spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, trehalose, sucrose, glycerol or sorbitol or a combination thereof and preferably magnesium sulfate.

[0229] In some examples, at least one of the reaction mixtures comprises glycerol at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1.5% or about 2% or about 2.5%. In some examples, at least one of the reaction mixtures comprises glucose at a concentration of between about 0.1 % and 10%, such as between about 0.5% and 10%, or between about 0.5% and 9%, or between about 0.5% and 8%, or between about 0.5% and 7%, or between about 0.5% and 6%, or between about 0.5% and 5%, or between about 0.5% and 4%, or between about 0.5% and 3%, or between about 0.5% and 2.5%, or between about 0.5% and 2%, such as about 0.5% or about 1 % or about 1 .5% or about 2% or about 2.5%.

[0230] In some examples, at least one of the reaction mixtures comprises a salt such as NFUCI or a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate. The magnesium salt (eg, MgCh) may be present at a concentration of between about 0.5 mM and 10 mM, or between about 1 mM and 5 mM, or between about 1 mM and 4 mM or between about 1 mM and 3mM, such as about 2 mM. The NFUCI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1 .5 mg / mL, such as about 1 mg / mL. The NH4CI may be present at a concentration of between about 0.5 mg / mL and 5 mg / mL, such as between about 0.5 mg / mL and 4 mg / mL, or between about 0.5 mg / mL and 3 mg / mL, or between about 0.5 mg / mL and 2 mg / mL, or between about 0.5 mg / mL and 1.5 mg / mL, such as about 1 mg / mL. In some examples, the reactionAU_Active01 27520873v1 CHRISTMmixture comprises a buffer such as a phosphate buffer. In some examples, the phosphate buffer comprises potassium phosphate or sodium phosphate. The buffer may be present at a concentration of between about 10 mM and 250 mM, such as between about 15 mM and 200 mM, or between about 20 mM and 150 mM, or between about 25 mM and 100 mM, or between about 25 mM and 75 mM, or between about 35 mM and 65 mM, or between about 40 mM and 60 mM, such as about 50 mM.

[0231] In some examples, the pH of at least one of the reaction mixtures is between about 5 and 9, such as about 5.5, about 6, about 6.5, about 7, about 7.5, about 8 or about 8.5. In some examples, the pH of at least one reaction mixture is between about 6 and 8.

[0232] In some examples, at least one of the reaction mixtures is incubated at a temperature of between about 15 °C and 45 °C, such as between about 15 °C and 40 °C, or between about 20 °C and 40 °C, or between about 20 °C and 37 °C, or between about 20 °C and 35 °C, or between about 20 °C and 30 °C, or between about 22.5 °C and 30 °C, or between about 22.5 °C and 27.5 °C. In some examples, the reaction mixture is incubated at a temperature of about 20 °C, or about 21 °C, or about 22 °C, or about 23 °C, or about 24 °C, or about 25 °C, or about 26 °C, or about 27 °C, or about 28 °C, or about 29 °C, or about 30 °C, or about 31 °C, or about 32 °C, or about 33 °C, or about 34 °C, or about 35 °C or about 36 °C, or about 37 °C. In some examples, at least one of the reaction mixtures is incubated at a temperature of about 25 °C.Pharmaceutical compositions and therapeutic applications

[0233] The present disclosure also provides pharmaceutical compositions comprising psilocybin, derivatives thereof and / or intermediates thereof produced by any of the methods described herein. The pharmaceutical composition may be suitable for administration nasally, orally, subcutaneously, parenterally or intravenously. Pharmaceutical compositions for oral use can be obtained by combining the bioactive agents with solid excipients and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present disclosure may be manufactured in a manner that is itself known, eg. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0234] The pharmaceutical compositions may be used to treat a neurological condition. The neurological condition to be treated may be disruptive mood dysregulation disorder, major depressive disorder, treatment-resistant depression, persistent depressive disorder, post-partum depression, an anxiety disorder, a substance-related disorder or post-traumatic stress disorder (PTSD). In some examples, the provides a method of treating a depressive disorder such as treatment-resistant depression. As used herein, the terms "depressive disorder", "depression disorder", or "depression" referAU_Active01 27520873v1 CHRISTMto a group of disorders characterized by low mood that can affect a person's thoughts, behaviour, feelings, and sense of well-being lasting for a period of time. The depressive disorder may disrupt the physical and psychological functions of a person, and in some examples, it causes a physical symptom such as weight loss, aches or pains, headaches, cramps, or digestive problems. The depressive disorder may cause a psychological symptom such as prolonged or persistent sadness, anxiety, a sense of hopelessness and irritability, feelings of guilt, worthlessness or helplessness, loss of interest or pleasure in hobbies, difficulty concentrating, remembering or making decisions. In some examples, the pharmaceutical composition is used to treat a mental health condition such as suicidality, depression, obsessive-compulsive disorder, anxiety, drug or alcohol dependence, tobacco dependence, cocaine- related disorders, cluster headache or post-traumatic stress disorder.

[0235] The terms "treat," "treating" or "treatment," as used herein, include alleviating, abating or ameliorating at least one symptom of a neurological condition, preventing additional symptoms, inhibiting the condition, e.g., arresting the development of the condition, relieving the condition, causing regression of the condition, delaying progression of the condition, or stopping the symptoms of the condition either prophylactically and / or therapeutically.

[0236] In some examples, the methods of the present disclosure reduce at least one symptom of depression. Symptoms of depression may include depressed mood, diminished interest in activities, weight loss or gain, decrease or increase in appetite, insomnia or hypersomnia, psychomotor agitation or retardation, fatigue or loss of energy, feelings of worthlessness or excessive or inappropriate guilt, diminished ability to concentrate or indecisiveness, or suicidal ideation or behaviour.Sequences

[0237] Table 3 lists various enzymes and peptides that are relevant to the present disclosure.Table 3. Enzymes and peptides relevant to the present disclosureNote: L-tryptophan decarboxylase (PsiD) Genbank identifier: ASU62239.1

[0238] Table 4 provides the sequences of the enzymes and peptides listed in Table 3.AU_Active01 27520873v1 CHRISTMTable 4. Sequences relevant to the present disclosureItems of the present disclosure

[0239] Set forth below are non-limiting Items of the present disclosure.Item 1 . A method of producing psilocybin comprising:AU_ActiveO1 27520873v1 CHRISTM(a) preparing a reaction mixture comprising tryptamine, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase, a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4- hydroxytryptamine, the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin, the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.Item 2. The method of Item 1 wherein the monooxygenase is a cytochrome P450 monooxygenase.Item 3. The method of Item 1 or Item 2 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.Item 4. The method of any one of Items 1 to 3 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.Item 5. The method of any one of Items 1 to 4 wherein the first strain is engineered to express a reductase.Item 6. The method of Item 5 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.Item 7. The method of Item 5 or Item 6 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.Item 8. The method of any one of Items 5 to 7 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.Item 9. The method of any one of Items 5 to 8 wherein: the monooxygenase and / or the reductase are encoded by nucleic acids codon optimised for expression in the first strain; the kinase is encoded by a nucleic acid codon optimised for expression in the second strain; and / or the methyltransferase is encoded by a nucleic acid codon optimised for expression in the third strain.Item 10. The method of any one of Items 1 to 9 wherein the first strain of cells has been cultured in the presence of b-aminolevulinic acid.Item 11 . The method of any one of Items 1 to 9 wherein the first strain of cells has been cultured in the absence of b-aminolevulinic acid.AU_Active01 27520873v1 CHRISTMItem 12. The method of any one of Items 1 to 11 wherein the first strain of cells has been cultured in a medium comprising soytone.Item 13. The method of any one of Items 1 to 12 wherein the kinase is a Psilocybe cubensis 4- hydroxytryptamine kinase or a variant or derivative thereof.Item 14. The method of any one of Items 1 to 13 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.Item 15. The method of any one of Items 1 to 14 wherein the methyltransferase is a / V-methyltransferase.Item 16. The method of any one of Items 1 to 15 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.Item 17. The method of any one of Items 1 to 16 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.Item 18. The method of any one of Items 1 to 17 wherein the reaction mixture further comprises S- adenosylmethionine (SAM) which provides a methyl for the methylation reactions.Item 19. The method of any one of Items 1 to 18 wherein the reaction mixture further comprises adenosine triphosphate.Item 20. The method of any one of Items 1 to 19 wherein the reaction mixture further comprises methionine, preferably L-methionine.Item 21 . The method of any one of Items 1 to 20 wherein the reaction mixture further comprises glycerol or glucose.Item 22. The method of any one of Items 1 to 21 wherein the reaction mixture further comprises a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate.Item 23. The method of any one of Items 1 to 22 wherein the reaction mixture comprises intact cells of the first strain, intact cells of the second strain and / or intact cells of the third strain.Item 24. The method of any one of Items 1 to 23 wherein the first strain, the second strain and / or the third strain carry a defective or suppressed tryptophanase.Item 25. The method of any one of Items 1 to 24 wherein the reaction mixture further comprises a membrane permeabilising agent.Item 26. The method of Item 25 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.Item 27. The method of any one of the preceding Items wherein the first strain, the second strain and the third strain are different strains of microorganisms.Item 28. The method of any one of the preceding Items wherein the first strain, the second strain and the third strain are different strains of E. coll.Item 29. The method of any one of the preceding Items wherein preparing the reaction mixture comprises separately adding the first strain, the second strain and the third strain to the reaction mixture.AU_Active01 27520873v1 CHRISTMItem 30. The method of any one of the preceding Items wherein the first strain, the second strain and the third strain are separately cultured before each strain is added to the reaction mixture.Item 31. The method of any one of the preceding Items wherein the second strain does not comprise: a Psilocybe cubensis methyltransferase or a variant or derivative thereof; and / or a tryptophan decarboxylase.Item 32. The method of any one of the preceding Items wherein the third strain does not comprise: a 4-hydroxytryptamine kinase; and / or a tryptophan decarboxylase.Item 33. The method of any one of the preceding Items wherein the first strain of cells, the second strain of cells and / or the third strain of cells has been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 34. The method of Item 33 wherein the first strain of cells, the second strain of cells and / or the third strain of cells has been spray dried, freeze dried, air dried or vacuum dried.Item 35. The method of Item 33 or Item 34 wherein the first strain of cells, the second strain of cells and / or the third strain of cells has been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.Item 36. A method of producing 4-hydroxytryptamine comprising: preparing a reaction mixture comprising tryptamine, and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase; and incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine.Item 37. The method of Item 36 wherein the monooxygenase is a cytochrome P450 monooxygenase.Item 38. The method of Item 36 or Item 37 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.Item 39. The method of any one of Items 36 to 38 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.Item 40. The method of any one of Items 36 to 39 wherein the cells are engineered to express a reductase.Item 41. The method of Item 40 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.Item 42. The method of Item 40 or Item 41 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.Item 43. The method of any one of Items 40 to 42 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.AU_Active01 27520873v1 CHRISTMItem 44. The method of any one of Items 40 to 43 wherein the monooxygenase and the reductase are encoded by a nucleic acid codon optimised for expression in the cells.Item 45. The method of any one of Items 36 to 44 wherein the reaction mixture comprises intact cells.Item 46. The method of any one of Items 36 to 45 wherein the cells carry a defective or suppressed tryptophanase.Item 47. The method of any one of Items 36 to 46 wherein the reaction mixture further comprises a membrane permeabilising agent.Item 48. The method of Item 47 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.Item 49. The method of any one of Items 36 to 48 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 50. The method of Item 49 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.Item 51 . The method of Item 40 or Item 50 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.Item 52. A method of producing norbaeocystin comprising: preparing a reaction mixture comprising4-hydroxytryptamine, and cells or a lysate thereof wherein the cells are engineered to express a kinase; and incubating the reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.Item 53. The method of Item 52 wherein the kinase is a 4-hydroxytryptamine kinase.Item 54. The method of Item 52 or Item 53 wherein the kinase is a Psilocybe cubensis 4- hydroxytryptamine kinase or a variant or derivative thereof.Item 55. The method of any one of Items 52 to 54 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.Item 56. The method of any one of Items 52 to 55 wherein the kinase is encoded by a nucleic acid codon optimised for expression in the cells.Item 57. The method of any one of Items 52 to 56 wherein the reaction mixture further comprises adenosine triphosphate.Item 58. The method of any one of Items 52 to 57 wherein the reaction mixture further comprises glycerol or glucose.Item 59. The method of any one of Items 52 to 58 wherein the reaction mixture further comprises a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate.AU_Active01 27520873v1 CHRISTMItem 60. The method of any one of Items 52 to 59 wherein the reaction mixture comprises intact cells.Item 61 . The method of any one of Items 52 to 60 wherein the cells carry a defective or suppressed tryptophanase.Item 62. The method of any one of Items 52 to 61 wherein the reaction mixture further comprises a membrane permeabilising agent.Item 63. The method of Item 62 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.Item 64. The method of any one of Items 52 to 63 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 65. The method of Item 64 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.Item 66. The method of Item 64 or Item 65 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.Item 67. The method of any one of Items 52 to 66 wherein the reaction mixture further comprises tryptamine, and cells engineered to express a monooxygenase and a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine to produce the 4- hydroxytryptamine; and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.Item 68. The method of any one of Items 52 to 66 wherein the 4-hydroxytryptamine is produced by the method of any one of Items 36 to 51 .Item 69. A method of producing psilocybin comprising: preparing a reaction mixture comprising norbaeocystin, and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase; and incubating the reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.Item 70. The method of Item 69 wherein the methyltransferase is a / V-methyltransferase.Item 71. The method of Item 69 or Item 70 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.Item 72. The method of any one of Items 69 to 71 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.AU_Active01 27520873v1 CHRISTMItem 73. The method of any one of Items 69 to 72 wherein the reaction mixture further comprises S- adenosylmethionine which provides a methyl for the methylation reactions.Item 74. The method of any one of Items 69 to 73 wherein the reaction mixture further comprises adenosine triphosphate and L-methionine to facilitate the generation of S-adenosylmethionine.Item 75. The method of any one of Items 69 to 74 wherein the methyltransferase is encoded by a nucleic acid codon optimised for expression in the cells.Item 76. The method of any one of Items 69 to 75 wherein the reaction mixture comprises intact cells.Item 77. The method of any one of Items 69 to 76 wherein the cells carry a defective or suppressed tryptophanase.Item 78. The method of any one of Items 69 to 77 wherein the reaction mixture further comprises methionine, preferably L-methionine.Item 79. The method of any one of Items 69 to 78 wherein the reaction mixture further comprises a membrane permeabilising agent.Item 80. The method of Item 79 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.Item 81 . The method of any one of Items 69 to 80 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 82. The method of Item 81 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.Item 83. The method of Item 81 or Item 82 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.Item 84. The method of any one of Items 69 to 83 wherein the reaction mixture further comprises 4- hydroxytryptamine, and cells engineered to express a kinase or a lysate thereof and wherein the reaction mixture is incubated under conditions suitable for: the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce the norbaeocystin; the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin; and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.Item 85. The method of any one of Items 69 to 83 wherein the norbaeocystin is produced by a method of any one of Items 52 to 68.Item 86. The method of any one of Items 69 to 85 wherein the reaction mixture further comprises tryptamine, and cells engineered to express a monooxygenase and a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine to produce the 4- hydroxytryptamine; the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin; the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin; andAU_Active01 27520873v1 CHRISTMthe methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.Item 87. The method of any one of Items 69 to 85 wherein the 4-hydroxytryptamine is produced according to any one of Items 36 to 51 .Item 88. A method of producing psilocybin comprising:(a) preparing a first reaction mixture comprising tryptamine, and a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase,(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine;(c) preparing a second reaction mixture by combining the 4-hydroxytryptamine with a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin, the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.Item 89. A method of producing psilocybin comprising:(a) preparing a first reaction mixture comprising tryptamine, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase, and a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase,(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4- hydroxytryptamine, and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin,(c) preparing a second reaction mixture by combining the norbaeocystin with a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.AU_ActiveO1 27520873v1 CHRISTMItem 90. The method of Item 88 or Item 89 wherein the first reaction mixture and / or the second reaction mixture further comprises a membrane permeabilising agent.Item 91 . The method of Item 91 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.Item 92. The method of any one of Items 88 to 91 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been dried or powderised and optionally rehydrated before preparing the first and / or second reaction mixture.Item 93. The method of Item 92 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried.Item 94. The method of Item 92 or Item 93 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, glycerol, trehalose or sorbitol and preferably magnesium sulfate.Item 95. A system for producing psilocybin comprising: tryptamine; a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase; a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase; and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase, wherein: the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4- hydroxytryptamine; the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin; the methyltransferase is capable of catalysing methylation of the norbaeocystin to produce baeocystin; and the methyltransferase is capable of catalysing methylation of the baeocystin to produce psilocybin.Item 96. The system of Item 95 wherein the monooxygenase is a cytochrome P450 monooxygenase.Item 97. The system of Item 95 or Item 96 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.Item 98. The system of any one of Items 95 to 97 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.Item 99. The system of any one of Items 95 to 98 wherein the first strain is engineered to express a reductase.Item 100. The system of Item 99 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.AU_Active01 27520873v1 CHRISTMItem 101 . The system of Item 99 or Item 100 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.Item 102. The system of any one of Items 99 to 101 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.Item 103. The system of any one of Items 99 to 102 wherein: the monooxygenase and / or the reductase are encoded by nucleic acids codon optimised for expression in the first strain; the kinase is encoded by a nucleic acid codon optimised for expression in the second strain; and / or the methyltransferase is encoded by a nucleic acid codon optimised for expression in the third strain.Item 104. The system of any one of Items 95 to 103 wherein the first strain of cells has been cultured in the presence of b-aminolevulinic acid.Item 105. The system of any one of Items 95 to 103 wherein the first strain of cells has been cultured in the absence of b-aminolevulinic acid.Item 106. The system of any one of Items 95 to 105 wherein the first strain of cells has been cultured in a medium comprising soytone.Item 107. The system of any one of Items 95 to 106 wherein the kinase is a Psilocybe cubensis 4- hydroxytryptamine kinase or a variant or derivative thereof.Item 108. The system of any one of Items 95 to 107 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.Item 109. The system of any one of Items 95 to 108 wherein the methyltransferase is a / V- methyltransferase.Item 110. The system of any one of Items 95 to 109 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.Item 11 1. The system of any one of Items 95 to 1 10 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.Item 112. The system of any one of Items 95 to 1 11 further comprising S-adenosylmethionine (SAM).Item 113. The system of any one of Items 95 to 112 further comprising adenosine triphosphate and L- methionine.Item 114. The system of any one of Items 95 to 113 comprising intact cells of the first strain, intact cells of the second strain and / or intact cells of the third strain.Item 115. The system of any one of Items 95 to 114 wherein the first strain, the second strain and / or the third strain carry a defective or suppressed tryptophanase.Item 116. The system of any one of Items 95 to 115 further comprising a membrane permeabilising agent.AU_Active01 27520873v1 CHRISTMItem 117. The system of Item 116 wherein the permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.Item 118. The system of any one of Items 95 to 117 wherein the first strain, the second strain and the third strain are different strains of microorganisms.Item 119. The system of any one of Items 95 to 118 wherein the first strain, the second strain and the third strain are different strains of E. coli.Item 120. The system of any one of Items 95 to 119 wherein the second strain does not comprise: a Psilocybe cubensis methyltransferase or a variant or derivative thereof; and / or a tryptophan decarboxylase.Item 121 . The system of any one of Items 95 to 120 wherein the third strain does not comprise: a 4-hydroxytryptamine kinase; and / or a tryptophan decarboxylase.Item 122. The system of any one of Items 95 to 121 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 123. The system of Item 122 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried.Item 124. The system of Item 122 or Item 123 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.Item 125. A system for producing 4-hydroxytryptamine comprising: tryptamine; and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4- hydroxytryptamine.Item 126. The system of Item 125 wherein the monooxygenase is a cytochrome P450 monooxygenase.Item 127. The system of Item 125 or Item 126 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.Item 128. The system of any one of Items 125 to 127 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.Item 129. The system of any one of Items 125 to 128 wherein the cells are engineered to express a reductase.Item 130. The system of Item 129 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.Item 131 . The system of Item 129 or Item 130 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.AU_Active01 27520873v1 CHRISTMItem 132. The system of any one of Items 129 to 131 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.Item 133. The system of any one of Items 129 to 132 wherein the monooxygenase and the reductase are encoded by a nucleic acid codon optimised for expression in the cells.Item 134. The system of any one of Items 125 to 133 comprising intact cells.Item 135. The system of any one of Items 125 to 134 wherein the cells carry a defective or suppressed tryptophanase.Item 136. The system of any one of Items 125 to 135 further comprising a membrane permeabilising agent.Item 137. The system of any one of Items 125 to 136 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.Item 138. The system of any one of Items 125 to 137 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 139. The system of Item 138 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.Item 140. The system of Item 138 or Item 139 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, glycerol, trehalose or sorbitol and preferably magnesium sulfate.Item 141 . A system for producing norbaeocystin comprising:4-hydroxytryptamine, and cells or a lysate thereof wherein the cells are engineered to express a kinase, wherein the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.Item 142. The system of Item 141 wherein the kinase is a 4-hydroxytryptamine kinase.Item 143. The system of Item 141 or Item 142 wherein the kinase is a Psilocybe cubensis 4- hydroxytryptamine kinase or a variant or derivative thereof.Item 144. The system of any one of Items 141 to 143 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.Item 145. The system of any one of Items 141 to 144 wherein the kinase is encoded by a nucleic acid codon optimised for expression in the cells.Item 146. The system of any one of Items 141 to 145 comprising intact cells.Item 147. The system of any one of Items 141 to 146 wherein the cells carry a defective or suppressed tryptophanase.Item 148. The system of any one of Items 141 to 147 further comprising a membrane permeabilising agent.Item 149. The system of Item 148 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.AU_Active01 27520873v1 CHRISTMItem 150. The system of any one of Items 141 to 149 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 151. The system of Item 150 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.Item 152. The system of Item 150 or Item 151 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, glycerol, trehalose or sorbitol and preferably magnesium sulfate.Item 153. The system of any one of Items 141 to 152 further comprising: tryptamine; and cells engineered to express a monooxygenase and a reductase or a lysate thereof, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4-hydroxytryptamine.Item 154. A system for producing psilocybin comprising: norbaeocystin; and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase, wherein the methyltransferase is capable of catalysing: methylation of the norbaeocystin to produce baeocystin; and methylation of the baeocystin to produce psilocybin.Item 155. The system of Item 154 wherein the methyltransferase is a / V-methyltransferase.Item 156. The system of Item 154 or Item 155 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.Item 157. The system of any one of Items 154 to 156 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.Item 158. The system of any one of Items 154 to 157 further comprising comprises S- adenosylmethionine.Item 159. The system of any one of Items 154 to 158 further comprising adenosine triphosphate and L- methionine.Item 160. The system of any one of Items 154 to 159 wherein the methyltransferase is encoded by a nucleic acid codon optimised for expression in the cells.Item 161. The system of any one of Items 154 to 160 comprising intact cells.Item 162. The system of any one of Items 154 to 161 wherein the cells carry a defective or suppressed tryptophanase.Item 163. The system of any one of Items 154 to 162 further comprising a membrane permeabilising agent.Item 164. The system of Item 163 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.AU_Active01 27520873v1 CHRISTMItem 165. The system of any one of Items 154 to 164 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.Item 166. The system of Item 165 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.Item 167. The system of Item 165 or Item 166 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, glycerol, trehalose or sorbitol and preferably magnesium sulfate.Item 168. The system of any one of Items 154 to 167 further comprising:4-hydroxytryptamine; and cells engineered to express a kinase or a lysate thereof, wherein the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce the norbaeocystin.Item 169. The system of Item 168 further comprising: tryptamine; and cells engineered to express a monooxygenase and a reductase or a lysate thereof, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce the 4- hydroxytryptamine.ExamplesMaterials and methodsChemicals

[0240] The analytical standards were tryptamine (Sigma Aldrich, cat. No. 94739), 4-hydroxytryptamine (Sapphire Bioscience, cat. no. 55206-11-6), norbaeocystin (Biosynth, cat. no. XN181311), baeocystin (Biosynth, cat. no. XB181309), psilocybin (Biosynth, cat. no. P-7825) and psilocin (Biosynth, cat. no. P- 7800). Cofactors and media supplements, S-adenosylmethionine (SAM, cat. no. 3493138), adenosine triphosphate (ATP, cat. no. 34369078), L-methionine (cat. no. M9625), b-aminolevulinic acid (cat. no. 5451092), were purchased from Sigma-Aldrich, Australia except SAM-e (S-adenosylmethionine, HerbaDiet). All the other chemicals were of high quality available from local suppliers.Media

[0241] LB agar plates contained yeast extract (5 g / L), Bacto tryptone (10 g / L), NaCI (10 g / L), bacteriological agar (25 g / L) and 100 pg / L of kanamycin or ampicillin as required.

[0242] LB broth contained Bacto tryptone (10 g / L), yeast extract (5 g / L), NaCI (5 g / ) and 100 pg / ml of kanamycin or ampicillin as required.

[0243] Auto-induction medium contained soy peptone (20 g / L), yeast extract (10 g / L), glycerol (30 g / L), KH2PO4 (0.75 g / L), Na2HPO4(0.75 g / L), MgSO4.7H2O (0.62 g / L), NaCI (10 g / L), NH4CI (1.4 g / L), 0.5 mM b-aminolevulinic acid, Fe(lll) citrate (12.5 mg / L), MnCI2.7H2O (15 mg / L), CuCI2.2H2O (1.5 mg / L), Zn acetate (1 .3 mg / L), NaMo.2H2Q (2.5 mg / L), Boric acid (2.5 mg / L), CoCI2.6H20 (2.5 mg / L), Na2EDTA (1 .4 mg / L). pH 7.0.AU_Active01 27520873v1 CHRISTM

[0244] Phosphate buffered saline contained Na2HPO4(1.09 g / L), NaH2PO4(0.32 g / L), NaCI (9 g / L), pH 7.4.

[0245] Phosphate buffer contained Na2HPO4(1.09 g / L), NaH2PO4(0.32 g / L), pH 7.0.DNA constructs

[0246] Nucleotide sequences for the truncated cytochrome P450 monooxygenase (PSiH_trunc) coupled with the cytochrome P450 reductase (CPR_cub), 4-hydroxytryptamine kinase (PsiK) and the methyltransferase (PsiM) were optimised for Escherichia coli codon usage using the Integrated DNA Technologies (idtdna.com) codon optimization tool. Optimised sequences were synthesised by Twist Bioscience (PsiH_trunc / CPR), and Gene Universal (PsiK and PsiM) and individually cloned into the pCW and pET29b expression vectors, respectively.

[0247] A truncated variant of the wild-type cytochrome P450 monooxygenase (PsiH_trunc) was used in this work. In the truncated variant, the N-terminus hydrophobic transmembrane domain was removed, and the transmembrane domain was replaced with a hydrophilic motif. The cytochrome P450 monooxygenase variant, PsiH_trunc and its partner cytochrome P450 reductase (CPR) coding sequences form a single operon with independent ribosome binding sites for each coding sequence.Strains and plasmids

[0248] E. coli BL21 (DE3) (Promega), E. coli KRX (Brookhaven National Laboratories), E. coli KRX AtnaA (tryptophanase knockout strain) were used as production hosts. The pCW and pET29b were used for cloning and expression of recombinant enzymes. Expression plasmid relevant to the present disclosure are listed in Table 5.Table 5. E. coli expression plasmids.Analytical methods - UPLC

[0249] Psilocybin and pathway intermediates were positively identified via Waters Acquity UPLC coupled to 2998 PDA (photodiode) and a Qda (MS) detectors, comparing retention times and parent ion masses to authentic standards. The MS detector runs single ion recording (SIR) monitoring for each analyte in addition to a scan function searching an 85 to 300 m / z range over the 15-minute time window. The MS parameters: gain = 1 ; probe temperature = 600°C; and the capillary voltage in positive mode is 1 kV. The UPLC-UV / MS was equipped with a HILIC-Z column (Agilent Technologies, Poroshell 120, 150 mm x 3mm, 2.7 pm). The two mobile phases, hydrophilic solvent A and hydrophobic solvent B were comprised of 10 mM ammonium formate, pH 3.5, and 90% acetonitrile plus 10 mM ammonium formate, pH 3.5, respectively. Milli Q water was used in all solutions. After injection of 5 pl sample or standard, solvent B was run at 100% for 1 minute before switching to gradient elution with solvent A for 10 minutes (0 - 95%), followed by a 4-minute re-equilibration phase. The flow rate was fixed at 0.5 ml / min and UVAU_Active01 27520873v1 CHRISTMdetection was at 280 nm. The MS detector was operated in positive ionization mode for the relevant characteristic parent ion masses of tryptamine ([M+H]+ = 161 , 4-hydroxytryptamine ([M+H]+ = 177), norbaeocystin ([M+H]+ = 257), baeocystin ([M+H]+ = 271), psilocin ([M+H]+ =206) and psilocybin ([M+H]+ = 285).Enzyme expression

[0250] The coding sequences for the PsiH variant, PsiH_trunc and the complementary CPR were cloned into the pCW expression vector. The plasmid was transformed into E. coli KRXAtnaA.

[0251] The coding sequences for PsiK and PsiM were cloned individually into the pET29b expression vector. The resulting pET29b_PsiK and pET29b_psiM plasmids were transformed into E. coli KRXAtnaA and E. coli BL21 (DE3).ResultsFlask scale expression of psilocybin biosynthesis pathway enzymes

[0252] Starter cultures were inoculated from aliquots of glycerin stock cultures into auto-induction medium supplemented with 100 micrograms / ml ampicillin or 100 micrograms / ml kanamycin (as appropriate) and incubated at 25°C for 12 - 16 hours. E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8), E. coli KRXAtnaA / pET29b_PsiK (A7) and E. coli BL21 (DE3) / pET29b_PsiM (F6) were inoculated into separate flasks of auto-induction medium containing 3% glycerol and the appropriate antibiotic and incubated at 25°C with shaking at 200 rpm for 48 hours, 28 - 30 hours, and 28 - 30 hours, respectively. Delta-aminolevulinic acid (0.5 mM) was included in the auto-induction medium for the E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8) culture, and 8 mM L-methionine was included in the autoinduction medium for E. coli BL21 (DE3) / pET29b_PsiM (F6). The pH was controlled at pH 7 with a 28% solution of ammonia. The cells were harvested by centrifugation (7000 rpm x 30 min) and washed in phosphate-buffered saline, pH 7.4, and stored as a wet paste (wet weight = 10 g / L) at -20°C for use in the biocatalytic conversion of tryptamine to norbaeocystin and psilocybin.Production of psilocybin biosynthesis pathway enzymes by fermentation

[0253] Seed cultures of E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8), E. coli KRXAtnaA / pET29b_PsiK (A7) and E. coli BL21 (DE3) / pET29b_PsiM (F6) were prepared by inoculating from aliquots of glycerin stock cultures into separate flasks of 50 ml auto-induction medium containing 3% glycerol. The cultures were incubated for 18 hours at 25 °C at 200 rpm.

[0254] The seed cultures were used to inoculate three fermenters (seed inoculum = 2%) containing auto-induction medium containing 6% glycerol and 100 pg / ml of the appropriate antibiotic in batch mode (500 ml). *Delta-aminolevulinic acid (0.5 mM) was included in the auto-induction medium for the E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8) culture, and 8 mM L-methionine was included in the autoinduction medium for the PsiM strain. Antifoam (1 ml) was added to the medium at the start of the fermentation and the pH was controlled at pH 7 with a 28% solution of ammonia and temperature at 25 - 30°C (Table 6). After 28 - 30 hours, when the cultures had reached an ODeoonm = 80 - 90, the cells were harvested by centrifugation at 7000 rpm for 30 minutes and washed in phosphate-buffered saline, pH 7.4. The cell pellets (about 110 g wet cell weight / l) were stored at -20°C for later use in the bioconversion of tryptamine to norbaeocystin or psilocybin.AU_Active01 27520873v1 CHRISTMTable 6. Batch fermentation conditions. PsiH = E. coli KRX AtnaA / pCW_PsiH_trunc_CPR (F8); PsiK = E. coli KRX AtnaA / pET29b_PsiK (A8); PsiM = E. coli BL21 / pET29b_PsiM (F6); amp = ampicillin; kan = kanamycin; wm = volume of air sparged per unit volume of culture medium per minuteSubstitution of 5-aminolevulinic acid (and soy peptone) with soytone

[0255] Delta-aminolevulinic acid may be considered an expensive compound. Medium costs may therefore be reduced by avoiding the use of b-aminolevulinic acid. The auto-induction medium was modified by replacing Soypeptone and b-aminolevulinic acid with Soytone (BactoTMsoytone) for the growth and expression of the cytochrome P450 monooxygenase (PsiH_trunc_CPR), which is a tryptamine 4-hydroxylase.

[0256] E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8) was cultivated in 500 ml auto-induction medium plus b-aminolevulinic acid (to assist functional expression of cytochrome P450 monooxygenase) or in 500 ml auto-induction medium replacing soy peptone and b-aminolevulinic acid with soytone as a growth substrate and inexpensive substitute for b-aminolevulinic acid. The cells were harvested after 48 hours and washed with 100 mM phosphate buffer, pH7.4, and the PsiH_trunc_CPR activity tested in the following assays:

[0257] The reaction mix (50 ml) contained 1.5 g / l tryptamine, 1 % glycerol, 1 mg / ml NFUCI, 28% NH3 (100 pl). The pH of the assay mixture was adjusted to 7.5 with HCI. E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8; Batch code: 22052024; ODeoonm = 20) was added to initiate the reaction. The assay was incubated at 25°C, and the pH controlled between 7 - 7.5.

[0258] After incubating the PsiH_trunc_CPR assay for 20 hours, E. coli KRXAtnaA / pCW_PsiH_trunc_CPR grown on auto-induction medium with soytone produced 3.65 mM 4- hydroxytryptamine compared to 8.3 mM 4-hydroxytryptamine produced by E. coli KRXAtnaA / pCW_PsiH_trunc_CPR grown on auto-induction medium with b-amino-levulinic acid and soypeptone. Soytone is a viable alternative to b-aminolevulinic acid for the expression of active PsiH_trunc. Using BactoTMsoytone in the auto-induction medium saves over $20 / L compared to autoinduction medium supplemented with 0.5 mM b-amino-levulinic acid (Sigma Aldrich) (Table 7).AU_Active01 27520873v1 CHRISTMTable 1. Price comparison of 6-amino-levulinic acid or soytone in auto-induction medium.Additives

[0259] The effect of additives including non-ionic detergents, ionic detergents and polar solvents on the methyltransferase (PsiM) reaction was tested. The PsiM assay contained about 1 mM mM norbaeocystin, 0.5% glycerol, 1 mg / ml NH4CI, 5 mM SAM in 100 mM phosphate buffer. The pH was adjusted to pH 7.5. The following additives were added to the assay mixture:• 0.5% Tween 20 (non-ionic)• 0.5% Triton-X 100 (non-ionic)• 0.1 % deoxycholate (ionic detergent)• 0.1 % cetyltriammonium bromide (CTAB, ionic detergent)• 2% methanol (polar, protic solvent)• 2% iso-propanol (polar, protic solvent)• 5% dimethylsulfoxide (DMSO, polar, aprotic solvent)

[0260] Two negative controls were included, namely, assay mixture without additives, with and without cells. E. coli BL21 / pET29b_PsiM cells (ODeoonm = 30) were added to initiate the reaction. The assay was incubated at 25°C at 200 rpm for 48h, and the pH controlled between 7 - 7.5. The PsiM-containing cells were grown as previously described with the inclusion of 8 mM L-methionine.

[0261] The membrane permeabilising agents enhanced psilocybin production, with the non-ionic detergent, Tween 20 and the polar solvents, methanol and DMSO having the most significant effect compared to the other additives (Figure 3).One-pot conversion of tryptamine to norbaeocystin

[0262] Higher norbaeocystin titers were achieved when E. coli KRXAtnaA / pET29b_PsiK (A7) cells were pre-treated with Tween 20 before addition to the bioconversion. For the pre-treatment procedure, E. coli KRXAtnaA / pET29b_PsiK (A7) cells were suspended in the pre-treatment buffer containing 12.54 g / l K2HPO4, 2.31 g / l KH2PO4, 5% glycerol and 2% Tween 20. The cells were incubated at 25°C. After 2 hours, the cells were centrifuged and washed three times with 50 mM phosphate buffer.

[0263] The bioconversion mixture (30 ml) contained 2 g / l tryptamine, 1 % glycerol, 1 mg / ml NH4CI and 28% NH3 (100 pL) and the pH was adjusted to pH 7.5 with HCI. E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells (F8; ODeoonm = 30) were added to the reaction mix to initiate the reaction and incubated at 25°C and shaken at 200 rpm. The pH was controlled between 7 - 7.5. After 30 hours, the temperature was increased to 37°C and pre-treated E. coli KRXAtnaA / pET29b_PsiK cells (A7, ODeoonm = 20) and 5 mM MgCh were added to the reaction. The reaction was incubated for another 10 hours.

[0264] After 30 hours, over 90% of the tryptamine was converted to 4-hydroxytryptamine by PsiH_trunc_CPR. (Figure 4). After the addition of pre-treated E. coli KRXAtnaA / pET29b_PsiK (A7) cells, 4.78 mM (1 .22 g / L) norbaeocystin was produced in 10 hours (30 - 40 hours).AU_Active01 27520873v1 CHRISTMOne-pot and two-pot bioconversion of tryptamine to psilocybin

[0265] A one-pot and a two-pot bioconversion of tryptamine to psilocybin was performed by sequentially adding E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8), E. coli KRXAtnaA / pET29b_PsiK (A7) and E. coli BL21 / pET29b_PsiM to the reaction mixture. In the case of the two-pot bioconversion, the biomass was removed from the reaction mix by centrifugation before the addition of E. coli BL21 / pET29b_PsiM (F6) cells. E. coli KRXAtnaA / pET29b_PsiK (A7) and E. coli BL21 / pET29b_PsiM (F6) were pre-treated with Tween 20 before adding to the reaction mixtures.One-pot bioconversion of tryptamine to psilocybin

[0266] In a pre-treatment stage, E. coli KRXAtnaA / pET29b_PsiK (A7) or E. coli BL21 / pET29b_PsiM (F6) cells were suspended in the pre-treatment buffer containing 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, 5% glycerol and 2% Tween 20 at pH 7.5. The cells were incubated at 25°C. After 2 hours, the cells were centrifuged and washed three times with 50 mM phosphate buffer (pH 7.5) before being used in the bioconversion.

[0267] The reaction mixture (50 ml) contained 0.7 g / L tryptamine, 0.5% glycerol, 1 mg / ml NH4CI and 28% NH3 (100 pl). After the pH was adjusted to pH 7.5 with HCI. E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8; ODeoonm = 20; from 6 L fermentation) was added to the reaction mixture to initiate the hydroxylation reaction. The reaction was incubated at 25°C and shaken at 200 rpm. After 20 hours, 0.5% glycerol, 1 mg / ml NH4CI, 28% NH3 (100 pl) and 5 mM MgCh were added to the mixture, the temperature increased to 37°C, and pre-treated E. coli KRXAtnaA / pET29b_PsiK (A7; OD600nm = 30 = 35g / L wet cell weight; from 6 L fermentation) and 10 mM MgCh were added to initiate the phosphorylation reaction. After 10 hours the temperature was shifted to 25°C, 0.5% glycerol, 1 mg / ml NH4CI, 28% NH3 (100 pl), and 5 mM SAM-e were added to the mixture, and pre-treated E. coli BL21 / pET29b_PsiM (F6; ODeoonm = 30 = 35 g / L; from 1 L fermentation with 8mM L-methionine added during fermentation) to initiate the methylation reactions. The reaction was incubated for a further 10 hours. The pH was controlled between pH 7 - 7.5 over the whole reaction.

[0268] After 20 h reaction time, 3.94 mM 4-hydroxytryptamine was produced from tryptamine by the cytochrome P450 monooxygenase (PsiH_trunc_CPR) (Figure 5). At this point, the temperature was increased to 37°C and E. coli KRXAtnaA / pET29b_PsiK containing 4-hydroxytryptamine kinase (PsiK) enzyme was added to the reaction mix. After 30 hours, tryptamine concentration was 1.11 mM, 4- hydroxytryptamine concentration was zero, and 1 .54 mM norbaeocystin was produced. The temperature was then shifted back to 25°C, and E. coli BL21 / pET29b_PsiM containing norbaeocystin methyltransferase (PsiM) was added to the bioconversion. After an additional 4 hours incubation, 0.81 mM psilocybin was produced which is equivalent to 230 mg / L psilocybin.One-pot bioconversion of tryptamine to psilocybin in a fermenter

[0269] In a pre-treatment stage, E. coli KRXAtnaA / pET29b_PsiK (A7) or E. coli BL21 / pET29b_PsiM (F6) cells were suspended in the pre-treatment buffer containing 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, 5% glycerol and 2% Tween 20 at pH 7.5. The cells were incubated at 25°C. After 2 hours, the cells were centrifuged and washed three times with 50 mM phosphate buffer (pH 7.5) before being used in the bioconversion.AU_Active01 27520873v1 CHRISTM

[0270] The reaction mixture (0.5 L) contained 1.5 g / L tryptamine, 0.5% glycerol and 1 mg / ml NH4CI. After the pH was adjusted to pH 7.5 with HCI, E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8; ODeoonm = 20; from 6 L fermentation) was added to the reaction mixture to initiate the hydroxylation reaction. The reaction was incubated at 25°C, and the dissolved oxygen was regulated at 30% by stirring the mixture at 400 - 1200 rpm and using a gas flow of 0 - 0.1 VVM (litre of air / liter medium / minute). After 30 hours, 0.5% glycerol, 1 mg / ml NH4CI and 10 mM MgCh were added to the mixture, the temperature increased to 37°C, and E. coli KRXAtnaA / pET29b_PsiK (A7; ODeoonm = 30; from 6 L fermentation) was added to initiate the phosphorylation reaction. The dissolved oxygen was regulated at 30% by stirring the mixture at 800 - 1200 rpm and using a gas flow of 0 - 0.2 VVM. After 4 hours the temperature was shifted to 28°C, 0.5% glycerol, 1 mg / ml NH4CI, 5 mM SAM-e were added to the mixture, and E. coli BL21 / pET29b_PsiM (F6; ODeoonm = 30; from 1 L fermentation with 8mM L-methionine added during fermentation) was added to initiate the methylation reactions. The dissolved oxygen was regulated at 30% by stirring the mixture at 800 - 1200 rpm and using a gas flow of 0 - 0.2 VVM. The reaction was incubated for a further 6 hours. The pH was controlled between pH 7 - 7.5 over the whole reaction with a mixture of 28% NH3 and 15% HCI.

[0271] After 30 h reaction time, 6.73 mM 4-hydroxytryptamine was produced from tryptamine by the cytochrome P450 monooxygenase (PsiH_trunc_CPR) (Figure 6). At this point, the temperature was increased to 37°C and 4-hydroxytryptamine kinase (PsiK) was added to the reaction mix. After 4 hours, 2.35 mM norbaeocystin was produced. The temperature was then shifted to 28°C, and norbaeocystin methyltransferase (PsiM) was added to the bioconversion. After an additional 2 hours incubation, 1.46 mM psilocybin was produced which is equivalent to 415 mg / L psilocybin.Two-pot bioconversion of tryptamine to psilocybin

[0272] The reaction mixture (50 ml) contained 1 .5 g / L tryptamine, 1 .0% glycerol, 1 mg / ml NH4CI and 28% NH3 (100 pl). After the pH was adjusted to pH 7.5 with HCI, E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8; ODeoonm = 20; from 6 L fermentation) was added to the reaction mixture to initiate the hydroxylation reaction. The reaction was incubated at 25°C and shaken at 200 rpm. After 40 hours, 0.5% glycerol, 1 mg / ml NH4CI, 28% NH3 (100 pl) and 5 mM MgCh were added to the mixture, the temperature increased to 37°C, and E. coli KRXAtnaA / pET29b_PsiK (A7; ODeoonm = 30; from 6 L fermentation) and 10 mM MgCh were added to initiate the phosphorylation reaction. After 10 hours, the biomass was removed by centrifugation, the temperature of the filtrate was adjusted to 25°C, 0.5% glycerol, 1 mg / ml NH4CI, 28% NH3 (100 pl), and 5 mM SAM-e were added to the mixture, and E. coli BL21 / pET29b_PsiM (F6; ODeoonm = 30; from 1 L fermentation with 8mM L-methionine added during fermentation) was added to initiate the methylation reactions. The reaction was incubated for a further 10 hours. The pH was controlled between pH 7 - 7.5 over the whole reaction.

[0273] After 40 h at 25°C, 13.34 mM 4-hydroxytryptamine was produced from tryptamine by the cytochrome P450 monooxygenase (PsiH_trunc_CPR) (Figure 7). At this stage, the temperature was increased to 37°C and 4-hydroxytryptamine kinase (PsiK) was added to the reaction mix. After 10 hours, 3.99 mM norbaeocystin (1 .02 g / L) was produced. The biomass was removed from the reaction mix and the temperature of the filtrate was adjusted to 25°C, norbaeocystin methyltransferase (PsiM) was addedAU_Active01 27520873v1 CHRISTMto the bioconversion, and the reaction mixture was incubated for a further 4 hours. At this time, 2.00 mM psilocybin was produced which is equivalent to 568 mg / L psilocybin.Freeze-drying E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8) biomass without additives

[0274] A drying experiment E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8) without the addition of protectants was carried out. Freshly harvested E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells (OD600nm = 100; 10 ml) cells were kept at -80°C for 4 - 8 hours. These cells were then dried in a VirTis BenchTop Pro with Omnitronics 8L BTP-8ZL00W freeze-drier for 2 days. Directly after drying E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8), the activity of these cells was tested.

[0275] Freeze-dried cells (10 mg dried cells / ml of rehydration buffer) were rehydrated in 100 mM phosphate buffer pH 7.5 with 5% glycerol 20 for 2 hours at 25 °C. After 2 hours of rehydration, the cells were harvested by centrifugation and washed with 100 mM phosphate buffer.

[0276] The assay contained 1.5 g / l tryptamine (9.4 mM), 1% glycerol, 1 mg / ml NH4CI and 28% NH3 (100 pL). The pH was adjusted to 7.5 with HCI. Re-hydrated E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8); equivalent to 10 mg dried cells / ml) was added to the assay to initiate the reaction. The assay was incubated at 25 °C and the pH was controlled between 7-7.5. No activity was detected indicating that the PsiH_trunc_CPR was inactivated during the drying process (Figure 8).Effect of sucrose, trehalose and sorbitol on the preservation of PsiH_trunc_CPR activity during freeze- drying

[0277] In this experiment, sucrose, trehalose and sorbitol were tested for their ability to protect PsiH_trunc_CPR activity during the drying process. Fresh E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells (ODeoonm = 100; 10 ml) were washed two time with 100 mM phosphate buffer, pH 7.4 and then resuspended in 5 ml buffer containing different stabilisers (2% w / v). The stabilisers tested were sucrose, sorbitol and trehalose (including a negative control without stabiliser). The cells were vortexed for 30 seconds and kept at -80°C for 4 - 8 hours. These cells were then dried in a VirTis BenchTop Pro with Omnitronics 8L BTP-8ZL00W freeze-drier for 2 days. Directly after drying E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8), the activity of these cells was tested.

[0278] Freeze-dried cells (10 mg dried cells / ml of rehydration buffer) were rehydrated in 100 mM phosphate buffer pH 7.5 with 5% glycerol 20 for 2 hours at 25 °C. After 2 hours of rehydration, the cells were harvested by centrifugation and washed with 100 mM phosphate buffer.

[0279] The assay contained 1.8 g / l tryptamine (11.25 mM), 1 % glycerol, 1 mg / ml NH4CI and 28% NH3 (100 pL). The pH was adjusted to 7.5 with HCI. Re-hydrated E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8); ODeoonm = 20) was added to the assay to initiate the reaction. The assay was incubated at 25 °C and the pH was controlled between 7-7.5.Effect of glucose, sucrose, trehalose and sorbitol in combination with MgSO4on the preservation of PsiH_trunc_CPR activity during freeze-drying

[0280] The effect of MgSC with glucose, sucrose, trehalose, sorbitol or sucrose plus glycerol on the preservation of cytochrome P450 monooxygenase (PsiH_trunc_CPR) during freeze-drying was tested.AU_Active01 27520873v1 CHRISTM

[0281] Fresh E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells (OD600nm = 100; 10 ml) direct from the fermenter were washed two times with 100 mM phosphate buffer, pH 7.4 and then resuspended in 5 ml of 25 mM MgSC plus different additives (2% w / v). The additives tested were glucose, sucrose, sorbitol, trehalose, or sucrose plus 0.5% glycerol. The cells were vortexed for 30 seconds and kept at -80°C for 4 - 8 hours. These cells were then dried in a VirTis BenchTop Pro with Omnitronics 8L BTP-8ZL00W freezedrier for 2 days. After drying, the activity of these cells was tested.

[0282] Freeze-dried cells (10 mg dried cells / ml of rehydration buffer) were rehydrated in 100 mM phosphate buffer pH 7.5 with 5% glycerol 20 for 2 hours at 25 °C. After 2 hours of rehydration, the cells were harvested by centrifugation and washed with 100 mM phosphate buffer.

[0283] The assay mix (100 ml) contained 1 .2 g / l tryptamine (7.5 mM), 1 % glycerol, 1 mg / ml NH4CI and 28% NH3 (100 pl). The pH was adjusted to 7.5 with HCI. Rehydrated E. coli KRXAtnaA / pCW_PsiH_trunc_CPR (F8) cells (ODeoonm = 20) were added to initiate the reactions. After 20 hours incubation, samples were analysed by TLC. After 20 hours, it was found that all the additives in combination with MgSCU except the combination of glucose and MgSC preserved enzyme activity during the drying process (Figure 9).Stability tests of dried E. coli KRXAtnaA / pCW_PsiH_trunc_CPR

[0284] A fresh batch of E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells were freeze-dried in the presence of 2% sorbitol + 25 mM MgSCU and stored at -20°C, 4°C and 25°C. The stability of the cytochrome P450 monooxygenase (tryptamine 4-hydroxylase) and partner cytochrome P450 reductase (PsiH_trunc_CPR) during storage was monitored for 1 month.

[0285] The dried E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells (10 mg dried cells / 1 ml of rehydration buffer) were rehydrated in 100 mM phosphate buffer, pH 7.5 containing 5% glycerol, for 2 hours at 25°C. After rehydration the cells were centrifuged and washed in 100 mM phosphate buffer, pH 7.4. The activity of the rehydrated biomass was then assayed.

[0286] The assay (30 ml) contained 7.5 mM tryptamine, 1 % glycerol, 1 mg / ml NH4CI, 28% NH3 (100 pl) in 100 mM phosphate buffer and the pH was adjusted to pH 7.5. Fresh or rehydrated E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells (equivalent of 10 mg dried cells / ml) were added to initiate the reaction. The assay was incubated at 25°C at 200 rpm, and the pH controlled between 7 - 7.5. Samples were analysed by UPLC. Cytochrome P450 monooxygenase (PsiH_trunc_CPR) in dried E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells was still active after 1 month storage at 4°C (Figure 10). However, after 2 weeks storage at 25°C, the same dried biomass retained only about 50% of its activity. The reduced activity of the dried PsiH_trunc_CPR-containing biomass measured at 25°C remained constant after 1 month storage at 25°C. In contrast, the activity of cytochrome P450 monooxygenase (tryptamine 4-hydroxylase) stored as wet E. coli KRXAtnaA / pCW_PsiH_trunc_CPR cells was inactivated in less than 2 weeks storage at 4°C and 25°C.Freeze-drying E. coli KRXAtnaA / pET29b_PsiK without additives

[0287] E. coli KRXAtnaA / pET29b_PsiK was freeze dried without additives. E. coli KRXAtnaA / pET29b_PsiK (ODeoonm = 90) cells direct from the fermenter were washed two times in 100 mM phosphate buffer, pH 7.4 and re-suspended in buffer without additives. The cells were vortexed forAU_Active01 27520873v1 CHRISTM30 seconds and kept at -80°C for 4 - 8 hours before drying in a VirTis BenchTop Pro with Omnitronics 8L BTP-8ZL00W freeze-drier for 2 days. Freeze-dried E. coli KRXAtnaA / pET29b_PsiK cells were stored at -20°C, 4°C and 25°C, and the activity of 4-hydroxytryptamine (PsiK) was determined at regular intervals.

[0288] The dried E. coli KRXAtnaA / pET29b_PsiK (10 mg dried cells / 1 ml of rehydration buffer) were rehydrated in 100 mM phosphate buffer, pH 7.5 containing 5% glycerol, 2% Tween 20 for 2 hours at 25°C. After rehydration the cells were centrifuged and washed in 100 mM phosphate buffer, pH 7.4.

[0289] The assay (5 ml) contained 2.8 mM 4-hydroxytryptamine, 1 % glycerol, 1 mg / ml NH4CI, 28% NH3 (100 pl) and 10 mM MgCh in 100 mM phosphate buffer and the pH was adjusted to pH 7.5. The rehydrated E. coli KRXAtnaA / pET29b_PsiK cells (equivalent of 10 mg dried cells / ml) were added to initiate the reaction. The assay was incubated at 25°C at 200 rpm, and the pH controlled between 7 - 7.5. Assay samples were analysed by UPLC.

[0290] The activity of 4-hydroxytryptamine kinase (PsiK) in dried E. coli KRXAtnaA / pET29b_PsiK cells was still stable after storing at 4°C for one month (Figure 11). 4-Hydroxytryptamine kinase (PsiK) activity in the same biomass stored at 25°C was stable for one week, but after 1 month storage retained only 45% of the original activity. In comparison, 4-hydroxytryptamine kinase (PsiK) in wet E. coli KRXAtnaA / pET29b_PsiK cells was stable after storing at 4°C and 25°C for one week but was inactivated after one month storage at these temperatures.Freeze-drying E. coli KRXAtnaA / pET29b_PsiK with additives

[0291] E. coli KRXAtnaA / pET29b_PsiK was freeze dried in the presence of 25 mM MgSCU, 25 mM MgSC + 2% sorbitol or 25 mM MgSCU + 2% trehalose. E. coli KRXAtnaA / pET29b_PsiK (ODeoonm = 90) cells direct from the fermenter were washed two times in 100 mM phosphate buffer, pH 7.4 and resuspended in buffer with additives. The cells were vortexed for 30 seconds and kept at -80°C for 4 - 8 hours before drying in a VirTis BenchTop Pro with Omnitronics 8L BTP-8ZL00W freeze-drier for 2 days. Freeze-dried E. coli KRXAtnaA / pET29b_PsiK cells were stored at 4°C and 25°C, and the activity of 4- hydroxytryptamine (PsiK) was determined at regular intervals.

[0292] The dried E. coli KRXAtnaA / pET29b_PsiK cells (10 mg) were rehydrated in 2 ml 100 mM phosphate buffer, pH 7.5 for 8 - 10 minutes at room temperature, vortexed and then centrifuged for 20 min. After centrifugation, the cells were separated and used directly in the assay.

[0293] The assay (4 ml) contained about 3 mM 4-hydroxytryptamine and 1 % glycerol in 100 mM phosphate buffer, and the pH was adjusted to pH 7.5. The rehydrated E. coli KRXAtnaA / pET29b_PsiK cells (equivalent of 10 mg dried cells / ml) were added to initiate the reaction. The assay was incubated at 25°C at 200 rpm, and the pH controlled manually between 7 - 7.5. Assay samples were analysed by UPLC.

[0294] The activity of 4-hydroxytryptamine kinase (PsiK) in dried E. coli KRXAtnaA / pET29b_PsiK cells was stable after storing at 4°C and 25°C for two weeks in the presence of the cryoprotectants MgSCU, and MgSC + sorbitol (Figure 13 & Figure 14), and after storing at 4°C for two weeks in the presence of the cryoprotectant MgSCU + trehalose (Figure15).Freeze-drying E. coli BL21 (DE3) / pET29b_PsiMAU_Active01 27520873v1 CHRISTM

[0295] E. coli BL21 (DE3) / pET29b_PsiM (ODeoonm = 90) cells direct from the fermenter were washed two times in 100 mM phosphate buffer, pH 7.4 and re-suspended in buffer without additives. The cells were vortexed for 30 seconds and kept at -80°C for 4 - 8 hours before drying in a VirTis BenchTop Pro with Omnitronics 8L BTP-8ZL00Wfreeze-drierfor2 days. Freeze-dried E. coli BL21 (DE3) / pET29b_PsiM cells were stored at 4°C and 25°C and the activity of norbaeocystin methyltransferase (PsiM) determined at regular intervals for one month.

[0296] The dried E. coli BL21 (DE3) / pET29b_PsiM cells (10 mg dried cells / 1 ml of rehydration buffer) were rehydrated in 100 mM phosphate buffer, pH 7.5 containing 5% glycerol, 2% Tween 20 for 2 hours at 25°C. After rehydration the cells were centrifuged and washed in 100 mM phosphate buffer, pH 7.4.

[0297] The assay (5 ml) contained 1.7 mM norbaeocystin, 1% glycerol, 1 mg / ml NH4CI, 28% NH3 (100 pl) and 8 mM SAM-e in 100 mM phosphate buffer and the pH was adjusted to pH 7.5. The rehydrated E. coli BL21 (DE3) / pET29b_PsiM cells (equivalent of 10 mg dried cells / ml) were added to initiate the reaction. The assay was incubated at 25°C and the pH controlled between 7 - 7.5. Assay samples were analysed by UPLC. The activity of norbaeocystin methyltransferase (PsiM) in dried E. coli BL21 (DE3) / pET29b_PsiM cells was stable at 4°C and 25°C for one week (Figure 12). After 1 month the activity of PsiM in dried E. coli BL21 (DE3) / pET29b_PsiM stored at 4°C and 25°C had reduced to about 40% of the original activity. The activity of norbaeocystin methyltransferase (PsiM) in wet E. coli BL21 (DE3) / pET29b_PsiM cells stored at 4°C for one week was stable, however, lost about 50% activity after one month. The norbaeocystin methyltransferase (PsiM) in the same wet biomass stored at 25°C was stable after 1 week storage but inactivated after one month storage.

[0298] It will be appreciated by those skilled in the art that the present disclosure may be embodied in many other forms.AU_Active01 27520873v1 CHRISTM

Claims

Claims1. A method of producing psilocybin comprising:(a) preparing a reaction mixture comprising tryptamine, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase, a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase; and(b) incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4- hydroxytryptamine, the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin, the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

2. The method of claim 1 wherein the monooxygenase is a cytochrome P450 monooxygenase.

3. The method of claim 1 or claim 2 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.

4. The method of any one of claims 1 to 3 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

5. The method of any one of claims 1 to 4 wherein the first strain is engineered to express a reductase.

6. The method of claim 5 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.AU_Active01 27520873v1 CHRISTM7. The method of claim 5 or claim 6 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.

8. The method of any one of claims 5 to 7 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.

9. The method of any one of claims 5 to 8 wherein: the monooxygenase and / or the reductase are encoded by nucleic acids codon optimised for expression in the first strain; the kinase is encoded by a nucleic acid codon optimised for expression in the second strain; and / or the methyltransferase is encoded by a nucleic acid codon optimised for expression in the third strain.

10. The method of any one of claims 1 to 9 wherein the first strain of cells has been cultured in the presence of b-aminolevulinic acid.11 . The method of any one of claims 1 to 9 wherein the first strain of cells has been cultured in the absence of b-aminolevulinic acid.

12. The method of any one of claims 1 to 1 1 wherein the first strain of cells has been cultured in a medium comprising soytone.

13. The method of any one of claims 1 to 12 wherein the kinase is a Psilocybe cubensis 4- hydroxytryptamine kinase or a variant or derivative thereof.

14. The method of any one of claims 1 to 13 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.

15. The method of any one of claims 1 to 14 wherein the methyltransferase is a / V-methyltransferase.AU_Active01 27520873v1 CHRISTM16. The method of any one of claims 1 to 15 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.

17. The method of any one of claims 1 to 16 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.

18. The method of any one of claims 1 to 17 wherein the reaction mixture further comprises S- adenosylmethionine (SAM) which provides a methyl for the methylation reactions.

19. The method of any one of claims 1 to 18 wherein the reaction mixture further comprises adenosine triphosphate.

20. The method of any one of claims 1 to 19 wherein the reaction mixture further comprises methionine, preferably L-methionine.21 . The method of any one of claims 1 to 20 wherein the reaction mixture further comprises glycerol or glucose.

22. The method of any one of claims 1 to 21 wherein the reaction mixture further comprises a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate.

23. The method of any one of claims 1 to 22 wherein the reaction mixture comprises intact cells of the first strain, intact cells of the second strain and / or intact cells of the third strain.

24. The method of any one of claims 1 to 23 wherein the first strain, the second strain and / or the third strain carry a defective or suppressed tryptophanase.AU_Active01 27520873v1 CHRISTM25. The method of any one of claims 1 to 24 wherein the reaction mixture further comprises a membrane permeabilising agent.

26. The method of claim 25 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

27. The method of any one of the preceding claims wherein the first strain, the second strain and the third strain are different strains of microorganisms.

28. The method of any one of the preceding claims wherein the first strain, the second strain and the third strain are different strains of E. coli.

29. The method of any one of the preceding claims wherein preparing the reaction mixture comprises separately adding the first strain, the second strain and the third strain to the reaction mixture.

30. The method of any one of the preceding claims wherein the first strain, the second strain and the third strain are separately cultured before each strain is added to the reaction mixture.31 . The method of any one of the preceding claims wherein the second strain does not comprise: a Psilocybe cubensis methyltransferase or a variant or derivative thereof; and / or a tryptophan decarboxylase.

32. The method of any one of the preceding claims wherein the third strain does not comprise: a 4-hydroxytryptamine kinase; and / or a tryptophan decarboxylase.

33. The method of any one of the preceding claims wherein the first strain of cells, the second strain of cells and / or the third strain of cells has been dried or powderised and optionally rehydrated before preparing the reaction mixture.AU_Active01 27520873v1 CHRISTM34. The method of claim 33 wherein the first strain of cells, the second strain of cells and / or the third strain of cells has been spray dried, freeze dried, air dried or vacuum dried.

35. The method of claim 33 or claim 34 wherein the first strain of cells, the second strain of cells and / or the third strain of cells has been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

36. A method of producing 4-hydroxytryptamine comprising: preparing a reaction mixture comprising tryptamine, and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase; and incubating the reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine.

37. The method of claim 36 wherein the monooxygenase is a cytochrome P450 monooxygenase.

38. The method of claim 36 or claim 37 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.

39. The method of any one of claims 36 to 38 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

40. The method of any one of claims 36 to 39 wherein the cells are engineered to express a reductase.

41. The method of claim 40 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.

42. The method of claim 40 or claim 41 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.AU_Active01 27520873v1 CHRISTM43. The method of any one of claims 40 to 42 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.

44. The method of any one of claims 40 to 43 wherein the monooxygenase and the reductase are encoded by a nucleic acid codon optimised for expression in the cells.

45. The method of any one of claims 36 to 44 wherein the reaction mixture comprises intact cells.

46. The method of any one of claims 36 to 45 wherein the cells carry a defective or suppressed tryptophanase.

47. The method of any one of claims 36 to 46 wherein the reaction mixture further comprises a membrane permeabilising agent.

48. The method of claim 47 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

49. The method of any one of claims 36 to 48 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.

50. The method of claim 49 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.51 . The method of claim 40 or claim 50 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.AU_Active01 27520873v1 CHRISTM52. A method of producing norbaeocystin comprising: preparing a reaction mixture comprising4-hydroxytryptamine, and cells or a lysate thereof wherein the cells are engineered to express a kinase; and incubating the reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.

53. The method of claim 52 wherein the kinase is a 4-hydroxytryptamine kinase.

54. The method of claim 52 or claim 53 wherein the kinase is a Psilocybe cubensis 4-hydroxytryptamine kinase or a variant or derivative thereof.

55. The method of any one of claims 52 to 54 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.

56. The method of any one of claims 52 to 55 wherein the kinase is encoded by a nucleic acid codon optimised for expression in the cells.

57. The method of any one of claims 52 to 56 wherein the reaction mixture further comprises adenosine triphosphate.

58. The method of any one of claims 52 to 57 wherein the reaction mixture further comprises glycerol or glucose.

59. The method of any one of claims 52 to 58 wherein the reaction mixture further comprises a magnesium salt, such as magnesium chloride, magnesium sulfate, magnesium citrate, magnesium sulphite, magnesium nitrite or magnesium nitrate.

60. The method of any one of claims 52 to 59 wherein the reaction mixture comprises intact cells.AU_Active01 27520873v1 CHRISTM61 . The method of any one of claims 52 to 60 wherein the cells carry a defective or suppressed tryptophanase.

62. The method of any one of claims 52 to 61 wherein the reaction mixture further comprises a membrane permeabilising agent.

63. The method of claim 62 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

64. The method of any one of claims 52 to 63 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.

65. The method of claim 64 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.

66. The method of claim 64 or claim 65 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

67. The method of any one of claims 52 to 66 wherein the reaction mixture further comprises tryptamine, and cells engineered to express a monooxygenase and a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine to produce the 4- hydroxytryptamine; and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.

68. The method of any one of claims 52 to 66 wherein the 4-hydroxytryptamine is produced by the method of any one of claims 36 to 51 .

69. A method of producing psilocybin comprising: preparing a reaction mixture comprisingAU_Active01 27520873v1 CHRISTMnorbaeocystin, and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase; and incubating the reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

70. The method of claim 69 wherein the methyltransferase is a / V-methyltransferase.

71. The method of claim 69 or claim 70 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.

72. The method of any one of claims 69 to 71 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.

73. The method of any one of claims 69 to 72 wherein the reaction mixture further comprises S- adenosylmethionine which provides a methyl for the methylation reactions.

74. The method of any one of claims 69 to 73 wherein the reaction mixture further comprises adenosine triphosphate and L-methionine to facilitate the generation of S-adenosylmethionine.

75. The method of any one of claims 69 to 74 wherein the methyltransferase is encoded by a nucleic acid codon optimised for expression in the cells.

76. The method of any one of claims 69 to 75 wherein the reaction mixture comprises intact cells.

77. The method of any one of claims 69 to 76 wherein the cells carry a defective or suppressed tryptophanase.AU_Active01 27520873v1 CHRISTM78. The method of any one of claims 69 to 77 wherein the reaction mixture further comprises methionine, preferably L-methionine.

79. The method of any one of claims 69 to 78 wherein the reaction mixture further comprises a membrane permeabilising agent.

80. The method of claim 79 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.81 . The method of any one of claims 69 to 80 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.

82. The method of claim 81 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.

83. The method of claim 81 or claim 82 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

84. The method of any one of claims 69 to 83 wherein the reaction mixture further comprises 4- hydroxytryptamine, and cells engineered to express a kinase or a lysate thereof and wherein the reaction mixture is incubated under conditions suitable for: the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce the norbaeocystin; the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin; and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

85. The method of any one of claims 69 to 83 wherein the norbaeocystin is produced by a method of any one of claims 52 to 68.AU_Active01 27520873v1 CHRISTM86. The method of any one of claims 69 to 85 wherein the reaction mixture further comprises tryptamine, and cells engineered to express a monooxygenase and a reductase or a lysate thereof, and wherein the reaction mixture is incubated under conditions suitable for: the monooxygenase to catalyse hydroxylation of the tryptamine to produce the 4- hydroxytryptamine; the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin; the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin; and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

87. The method of any one of claims 69 to 85 wherein the 4-hydroxytryptamine is produced according to any one of claims 36 to 51 .

88. A method of producing psilocybin comprising:(a) preparing a first reaction mixture comprising tryptamine, and a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase,(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4-hydroxytryptamine;(c) preparing a second reaction mixture by combining the 4-hydroxytryptamine with a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase, and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin, the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

89. A method of producing psilocybin comprising:(a) preparing a first reaction mixture comprising tryptamine, a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase and a reductase, and a second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase,AU_ActiveO1 27520873v1 CHRISTM(b) incubating the first reaction mixture under conditions suitable for the monooxygenase to catalyse hydroxylation of the tryptamine to produce 4- hydroxytryptamine, and the kinase to catalyse phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin,(c) preparing a second reaction mixture by combining the norbaeocystin with a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase;(d) incubating the second reaction mixture under conditions suitable for the methyltransferase to catalyse methylation of the norbaeocystin to produce baeocystin, and the methyltransferase to catalyse methylation of the baeocystin to produce psilocybin.

90. The method of claim 88 or claim 89 wherein the first reaction mixture and / or the second reaction mixture further comprises a membrane permeabilising agent.

91. The method of claim 91 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

92. The method of any one of claims 88 to 91 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been dried or powderised and optionally rehydrated before preparing the first and / or second reaction mixture.

93. The method of claim 92 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried.

94. The method of claim 92 or claim 93 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

95. A system for producing psilocybin comprising: tryptamine; a first strain of cells or a lysate thereof wherein the first strain is engineered to express a monooxygenase;AU_Active01 27520873v1 CHRISTMa second strain of cells or a lysate thereof wherein the second strain is engineered to express a kinase; and a third strain of cells or a lysate thereof wherein the third strain is engineered to express a methyltransferase, wherein: the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4- hydroxytryptamine; the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin; the methyltransferase is capable of catalysing methylation of the norbaeocystin to produce baeocystin; and the methyltransferase is capable of catalysing methylation of the baeocystin to produce psilocybin.

96. The system of claim 95 wherein the monooxygenase is a cytochrome P450 monooxygenase.

97. The system of claim 95 or claim 96 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.

98. The system of any one of claims 95 to 97 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

99. The system of any one of claims 95 to 98 wherein the first strain is engineered to express a reductase.

100. The system of claim 99 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.

101. The system of claim 99 or claim 100 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.

102. The system of any one of claims 99 to 101 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.AU_Active01 27520873v1 CHRISTM103. The system of any one of claims 99 to 102 wherein: the monooxygenase and / or the reductase are encoded by nucleic acids codon optimised for expression in the first strain; the kinase is encoded by a nucleic acid codon optimised for expression in the second strain; and / or the methyltransferase is encoded by a nucleic acid codon optimised for expression in the third strain.

104. The system of any one of claims 95 to 103 wherein the first strain of cells has been cultured in the presence of b-aminolevulinic acid.

105. The system of any one of claims 95 to 103 wherein the first strain of cells has been cultured in the absence of b-aminolevulinic acid.

106. The system of any one of claims 95 to 105 wherein the first strain of cells has been cultured in a medium comprising soytone.

107. The system of any one of claims 95 to 106 wherein the kinase is a Psilocybe cubensis 4- hydroxytryptamine kinase or a variant or derivative thereof.

108. The system of any one of claims 95 to 107 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.

109. The system of any one of claims 95 to 108 wherein the methyltransferase is a / V-methyltransferase.

110. The system of any one of claims 95 to 109 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.AU_Active01 27520873v1 CHRISTM111. The system of any one of claims 95 to 110 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.

112. The system of any one of claims 95 to 111 further comprising S-adenosylmethionine (SAM).

113. The system of any one of claims 95 to 112 further comprising adenosine triphosphate and L- methionine.

114. The system of any one of claims 95 to 113 comprising intact cells of the first strain, intact cells of the second strain and / or intact cells of the third strain.

115. The system of any one of claims 95 to 1 14 wherein the first strain, the second strain and / or the third strain carry a defective or suppressed tryptophanase.

116. The system of any one of claims 95 to 115 further comprising a membrane permeabilising agent.

117. The system of claim 1 16 wherein the permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

118. The system of any one of claims 95 to 117 wherein the first strain, the second strain and the third strain are different strains of microorganisms.

119. The system of any one of claims 95 to 118 wherein the first strain, the second strain and the third strain are different strains of E. coli.

120. The system of any one of claims 95 to 119 wherein the second strain does not comprise: a Psilocybe cubensis methyltransferase or a variant or derivative thereof; and / or a tryptophan decarboxylase.AU_Active01 27520873v1 CHRISTM121. The system of any one of claims 95 to 120 wherein the third strain does not comprise: a 4-hydroxytryptamine kinase; and / or a tryptophan decarboxylase.

122. The system of any one of claims 95 to 121 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.

123. The system of claim 122 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried.

124. The system of claim 122 or claim 123 wherein the first strain of cells, the second strain of cells and / or the third strain of cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

125. A system for producing 4-hydroxytryptamine comprising: tryptamine; and cells or a lysate thereof wherein the cells are engineered to express a monooxygenase, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce 4- hydroxytryptamine.

126. The system of claim 125 wherein the monooxygenase is a cytochrome P450 monooxygenase.

127. The system of claim 125 or claim 126 wherein the monooxygenase is a Psilocybe cubensis cytochrome P450 monooxygenase or a variant or derivative thereof.

128. The system of any one of claims 125 to 127 wherein the monooxygenase comprises the sequence set forth in SEQ ID NO: 2, or a sequence having at least about 80% identity to SEQ ID NO: 2.

129. The system of any one of claims 125 to 128 wherein the cells are engineered to express a reductase.AU_Active01 27520873v1 CHRISTM130. The system of claim 129 wherein the reductase is a Psilocybe cubensis cytochrome P450 reductase or a variant or derivative thereof.131 . The system of claim 129 or claim 130 wherein the reductase comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at least about 80% identity to SEQ ID NO: 3.

132. The system of any one of claims 129 to 131 wherein the monooxygenase and the reductase are encoded in a single operon comprising separate ribosome binding sites.

133. The system of any one of claims 129 to 132 wherein the monooxygenase and the reductase are encoded by a nucleic acid codon optimised for expression in the cells.

134. The system of any one of claims 125 to 133 comprising intact cells.

135. The system of any one of claims 125 to 134 wherein the cells carry a defective or suppressed tryptophanase.

136. The system of any one of claims 125 to 135 further comprising a membrane permeabilising agent.

137. The system of any one of claims 125 to 136 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

138. The system of any one of claims 125 to 137 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.

139. The system of claim 138 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.AU_Active01 27520873v1 CHRISTM140. The system of claim 138 or claim 139 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.141 . A system for producing norbaeocystin comprising:4-hydroxytryptamine, and cells or a lysate thereof wherein the cells are engineered to express a kinase, wherein the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce norbaeocystin.

142. The system of claim 141 wherein the kinase is a 4-hydroxytryptamine kinase.

143. The system of claim 141 or claim 142 wherein the kinase is a Psilocybe cubensis 4- hydroxytryptamine kinase or a variant or derivative thereof.

144. The system of any one of claims 141 to 143 wherein the kinase comprises the sequence set forth in SEQ ID NO: 4, or a sequence having at least about 80% identity to SEQ ID NO: 4.

145. The system of any one of claims 141 to 144 wherein the kinase is encoded by a nucleic acid codon optimised for expression in the cells.

146. The system of any one of claims 141 to 145 comprising intact cells.

147. The system of any one of claims 141 to 146 wherein the cells carry a defective or suppressed tryptophanase.

148. The system of any one of claims 141 to 147 further comprising a membrane permeabilising agent.AU_Active01 27520873v1 CHRISTM149. The system of claim 148 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

150. The system of any one of claims 141 to 149 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.151 . The system of claim 150 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.

152. The system of claim 150 or claim 151 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.

153. The system of any one of claims 141 to 152 further comprising: tryptamine; and cells engineered to express a monooxygenase and a reductase or a lysate thereof, wherein the monooxygenase is capable of catalysing hydroxylation ofthe tryptamine to produce 4-hydroxytryptamine.

154. A system for producing psilocybin comprising: norbaeocystin; and cells or a lysate thereof wherein the cells are engineered to express a methyltransferase, wherein the methyltransferase is capable of catalysing: methylation of the norbaeocystin to produce baeocystin; and methylation of the baeocystin to produce psilocybin.

155. The system of claim 154 wherein the methyltransferase is a / V-methyltransferase.

156. The system of claim 154 or claim 155 wherein the methyltransferase is a Psilocybe cubensis methyltransferase or a variant or derivative thereof.

157. The system of any one of claims 154 to 156 wherein the methyltransferase comprises the sequence set forth in SEQ ID NO: 5, or a sequence having at least about 80% identity to SEQ ID NO: 5.AU_Active01 27520873v1 CHRISTM158. The system of any one of claims 154 to 157 further comprising comprises S-adenosylmethionine.

159. The system of any one of claims 154 to 158 further comprising adenosine triphosphate and L- methionine.

160. The system of any one of claims 154 to 159 wherein the methyltransferase is encoded by a nucleic acid codon optimised for expression in the cells.

161. The system of any one of claims 154 to 160 comprising intact cells.

162. The system of any one of claims 154 to 161 wherein the cells carry a defective or suppressed tryptophanase.

163. The system of any one of claims 154 to 162 further comprising a membrane permeabilising agent.

164. The system of claim 163 wherein the membrane permeabilising agent is an alcohol, a polar protic solvent, a polar aprotic solvent or a surfactant.

165. The system of any one of claims 154 to 164 wherein the cells have been dried or powderised and optionally rehydrated before preparing the reaction mixture.

166. The system of claim 165 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried.

167. The system of claim 165 or claim 166 wherein the cells have been spray dried, freeze dried, air dried or vacuum dried with a stabiliser, such as glucose, sucrose, trehalose, glycerol or sorbitol and preferably magnesium sulfate.AU_Active01 27520873v1 CHRISTM168. The system of any one of claims 154 to 167 further comprising:4-hydroxytryptamine; and cells engineered to express a kinase or a lysate thereof, wherein the kinase is capable of catalysing phosphorylation of the 4-hydroxytryptamine to produce the norbaeocystin.

169. The system of claim 168 further comprising: tryptamine; and cells engineered to express a monooxygenase and a reductase or a lysate thereof, wherein the monooxygenase is capable of catalysing hydroxylation of the tryptamine to produce the 4- hydroxytryptamine.AU_ActiveO1 27520873v1 CHRISTM

Images