Method for producing tryptamine derivatives.

Abstract

The present disclosure provides a compound of formula (I): [Formula 1] TIFF2024520057000074.tif143159 wherein tryptamine derivative (I) is not tryptophan, 4-hydroxytryptamine, N-acetyl-4-hydroxytryptamine, norbaeocystin, baeocystin; psilocybin, psilocin, aeruginasin, halogenated tryptophan, halogenated tryptamine, halogenated N-methylated tryptamine, halogenated N,N-dimethyltryptamine or halogenated N,N,N-trimethyltryptamine; said method comprises producing a tryptamine derivative of formula (II): [Case 2] TIFF2024520057000075.tif158159 providing an indole acceptor of formula R II , R IV , R V , R VI or R VII is not H, and R III is H or CH2CH2NH2 or CH2CHCOOHNH2; and further comprising contacting said indole acceptor with a substituent donor in the presence of one or more enzymes that replace one or more of H, OH and / or COOH in said indole acceptor with one or more substituents of the substituent donor.

Description

[Technical field]

[0001] The present invention relates to a method for producing tryptamine derivatives and the tryptamine derivatives obtained therefrom. [Background technology]

[0002] Tryptamines are well known, including serotonin, an important neurotransmitter, and melatonin, a hormone involved in regulating the sleep-wake cycle. Tryptamine alkaloids are found in fungi, plants, and animals, and may also be used by humans for their neurological or psychotropic effects. Primary examples of tryptamine alkaloids include psilocybin (from the "psilocybin mushroom") and dimethyltryptamine (DMT). DMT is obtained from a number of plant sources, such as the chacruna, and is often used in ayahuasca beverages. Many synthetic tryptamines have also been produced, including migraine medications and hallucinogens. Substituted tryptamines, or serotonin analogs, are organic compounds that may be considered derived from tryptamine itself. The molecular structure of all tryptamines contains an indole ring attached to an amino (NH2) group via an ethyl (-CH2CH2-) side chain. In substituted tryptamines, the indole ring, side chain, and / or amino group are modified by replacing one of the hydrogen (H) atoms with another group.

[0003] WO2019173797 relates to microbial cells containing enzymes involved in the biosynthetic pathways converting anthranilic acid, indole or tryptophan to tryptamine. WO2021052989 relates to the biotechnological production of psilocybin in cell factories and to halogenated tryptamines.

[0004] There remains a continuing need to identify new substituted tryptamines having new and / or enhanced properties relating to the therapeutic efficacy, administration, formulation and / or manufacture of the substituted tryptamines and / or to devise new optimized methods for the manufacture of such substituted tryptamines. Summary of the Invention

[0005] The present invention provides certain improvements that provide solutions to the shortcomings of known substituted tryptamine derivatives and methods for their production using known techniques. Surprisingly, the present invention provides an enzyme that acts to catalyze the substitution of tryptamine both in vitro and in vivo, thereby avoiding the shortcomings of known techniques, and further provides an enzyme that is incorporated into genetically modified host cells and functions therein to produce such substituted tryptamine derivatives. The inventors have also found that the substitution of tryptamine not only produces previously unknown substituted tryptamine derivatives with interesting and useful properties, but that the in vivo expression of the substitution enzyme provides a series of previously unknown advantages in the process of producing substituted tryptamine derivatives in genetically modified cell factories such as yeast, including, but not limited to, (i) reduced product inhibition of the tryptamine production pathway due to the presence of high concentrations of tryptamine or tryptophan, (ii) enhanced export of compounds into the extracellular space, and (iii) reduced toxicity of tryptamine-derived compounds to microbial hosts.

[0006] Thus, in a first aspect, the present invention provides a compound of formula (I): [ka] wherein tryptamine derivative (I) is not tryptophan, 4-hydroxytryptamine, N-acetyl-4-hydroxytryptamine, norbaeocystin, baeocystin; psilocybin, psilocin, aeruginasin, halogenated tryptophan, halogenated tryptamine, halogenated N-methylated tryptamine, halogenated N,N-dimethyltryptamine or halogenated N,N,N-trimethyltryptamine; said method comprises producing a tryptamine derivative of formula (II): [ka] providing an indole acceptor of formula R II , R IV , RV , R VI or R VII is not H, and R III is H or CH2CH2NH2 or CH2CHCOOHNH2; and further, the method includes contacting the indole acceptor with a substituent donor in the presence of one or more enzymes that replace one or more of the H, OH and / or COOH in the indole acceptor with one or more substituents of the substituent donor.

[0007] In a further aspect, the present invention provides a compound of formula (I): [ka] wherein at least one of R2, R4 through R9, α and β is a glycosyl group. In particular, R2, R4 through R7, α and β may be an O-glycosyl group.

[0008] In a further aspect, the invention provides microbial host cells genetically engineered to carry out the methods of the invention and produce tryptamine derivatives, wherein the host cell expresses one or more heterologous genes encoding one or more enzymes which, in the presence of an indole acceptor and one or more substituent donors, transfer one or more substituents to one or more of the H, OH and / or COOH of the indole acceptor.

[0009] In further aspects, the present invention provides a cell culture medium comprising a host cell of the present invention and a growth medium; a fermentation medium comprising a tryptamine derivative (I) contained in the cell culture medium of the present invention; and a composition comprising a fermentation medium of the present invention and / or a tryptamine derivative (I) of the present invention and one or more drugs, additives and / or excipients. [Brief description of the drawings]

[0010] [Figure 1]1 shows the biosynthetic pathway for the production of tryptamine in Saccharomyces cerevisiae. Overexpressed genes are shown in bold and relevant gene deletions involved in the biosynthetic pathway are shown with a cross. [Diagram 2] A general scheme for the integration of gene overexpression cassettes into the Saccharomyces cerevisiae genome is shown. The linear expression cassettes contain overlapping homology to each other, while the outermost cassettes (Rec1 and Rec5) contain homology to the genomic landing pads of the Saccharomyces cerevisiae genome. Transformation of the linear cassettes results in assembly and integration by homologous recombination. [Diagram 3] shows gene deletion by URA3 marker replacement. The Ura3 cassette is amplified using primers that introduce upstream and downstream homology to the gene to be deleted. Gene deletion occurs by homologous recombination and replacement with the Ura3 marker. The marker can then be looped out in a scarless manner and the Ura3 marker can be reused. [Figure 4] 1 shows the structures of substituted tryptamine glucosides verified by LC-MS / QTOF. [Diagram 5] An example of an LC-MS-QTOF chromatogram from the in vitro conversion of psilocin to OBT-001 (psilocin-O-β-D-glucoside) by At71C2 (sequence numbers 193, 194) is shown, further showing the retention time (RT), predicted and measured masses of each compound, and fragmentation patterns determined by LC-MS / QTOF analysis. [Figure 6] Illustrated are example LC-MS-QTOF chromatograms from the in vitro conversion of noribogaine to OBT-002 (noribogaine-O-β-D-glucoside) by Pt73Y (sequence numbers 203, 204), further showing the retention time (RT), predicted and measured masses of each compound, and fragmentation patterns determined by LC-MS / QTOF analysis. [Figure 7]FIG. 1 shows an example of an LC-MS-QTOF chromatogram from the in vitro conversion of bufotenin to OBT-003 (bufotenin-O-β-D-glucoside) by At71C1_At71C2_353 (sequence numbers 243, 244), further showing the retention time (RT), predicted and measured masses of each compound, and fragmentation patterns determined by LC-MS / QTOF analysis. [Figure 8] Illustrated are example LC-MS-QTOF chromatograms from the in vitro conversion of serotonin to OBT-004 (serotonin-O-β-D-glucoside) by At71C1-Sr71E1_354 (sequence numbers 199, 200), further showing the retention time (RT), predicted and measured masses of each compound, and fragmentation patterns determined by LC-MS / QTOF analysis. [Figure 9] The structures of substituted tryptamine xylosides verified by LC-MS / QTOF are shown. [Figure 10] An example of an LC-MS-QTOF chromatogram from the in vitro conversion of psilocin to OBT-005 (psilocin-O-β-D-xyloside) by At71C2 (sequence numbers 193, 194) is shown, further showing the retention time (RT), predicted and measured masses of each compound, and fragmentation patterns determined by LC-MS / QTOF analysis. [Figure 11] Illustrated are example LC-MS-QTOF chromatograms from the in vitro conversion of noribogaine to OBT-006 (noribogaine-O-β-D-xyloside) by Pt73Y (sequence numbers 203, 204), further showing the retention time (RT), predicted and measured masses of each compound, and fragmentation patterns determined by LC-MS / QTOF analysis. [Figure 12] FIG. 1 shows an example of an LC-MS-QTOF chromatogram from the in vitro conversion of bufotenin to OBT-007 (bufotenin-O-β-D-xyloside) by Pt73Y (sequence numbers 203, 204), further showing the retention time (RT), predicted and measured masses of each compound, and fragmentation patterns determined by LC-MS / QTOF analysis. [Figure 13]1 shows the structure of psilocin di-glucoside produced by combining multiple glycosyltransferases OBT-008 (psilocin-O-β-D-glucoside-O-β-D-glucoside). [Figure 14] An example of tryptamine production in SC-62, a Saccharomyces cerevisiae strain genetically engineered to efficiently produce tryptamine, is shown. Shown is an HPLC chromatogram of SC-62 after cultivation compared to an authenticated tryptamine analytical standard. [Figure 15] An example of serotonin production in SC-75, a budding yeast strain genetically engineered to efficiently produce serotonin, is shown. Shown is an HPLC chromatogram of SC-75 after incubation compared to an authenticated serotonin analytical standard. [Figure 16] An example of 4-coumaroylserotonin production in ST-4CS2, a Saccharomyces cerevisiae strain genetically engineered to efficiently produce 4-coumaroylserotonin, is shown. Shown is an HPLC chromatogram of ST-4CS2 after cultivation compared to an authenticated 4-coumaroylserotonin analytical standard. [Figure 17] Shown is an example of psilocybin production in SC-206 and SC-302. SC-206 is a Saccharomyces cerevisiae strain genetically modified to produce psilocybin, and SC-302 is the same parent strain, but with the DIA3 gene knocked out. Shown is the HPLC chromatogram of SC-206 and SC-302 after cultivation compared to certified psilocybin analytical standard. [Figure 18] Shown are examples of psilocybin production in SC-275 and SC-268. SC-275 is a genetically modified Saccharomyces cerevisiae strain with a biosynthetic pathway derived from P.cubensis to produce psilocybin, and SC-268 is a strain with a biosynthetic pathway derived from P.cyanescens. Shown are HPLC chromatograms of SC-275 and SC-268 after cultivation compared to an authenticated psilocybin analytical standard. The inset shows a zoomed-in view of the psilocybin peak for clarity. [Figure 19]An example of melatonin production in SC-124, a budding yeast strain genetically modified to efficiently produce melatonin, is shown. Shown is an HPLC chromatogram of SC-124 after cultivation compared to an authenticated melatonin analytical standard. The inset shows a zoom in on the psilocybin peak for clarity.

[0011] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between the terminology in this specification and the incorporated references, the terminology set forth in this specification shall take precedence and control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] definition Any EC number that may be used herein refers to the Enzyme Nomenclature from NC-IUBMB, Academic Press, San Diego, California, 1992, including 30 Supplements 1-5, published in Eur.J.Bio-chem.1994,223,1-5; Eur.J.Biochem.1995,232,1-6; Eur.J.Biochem.1996,237,1-5; Eur.J.Biochem.1997,250,1-6; and Eur.J.Biochem.1999,264,610-650, respectively. The nomenclature is periodically supplemented and updated; see, for example, http: / / enzyme.expasy.org / .

[0013] As used herein, the term "fructose-6-phosphate phosphoketolase" refers to an enzyme that catalyzes the reaction of fructose-6-phosphate to erythrose-4-phosphate and acetyl phosphate.

[0014] As used herein, the term "phosphotransacetylase" refers to an enzyme that catalyzes the reaction of acetyl phosphate to acetyl-CoA.

[0015] As used herein, the term "DAHP synthase" refers to the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase enzyme, which catalyzes the reaction of phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP).

[0016] As used herein, the term "Aro1" refers to EPSP synthase, which catalyzes the conversion of DAHP to 5-enolpyruvoyl-shikimate 3-phosphate (EPSP).

[0017] As used herein, the term "shikimate kinase" refers to an enzyme that catalyzes the reaction of shikimic acid to shikimate-3-phosphate.

[0018] As used herein, the term "chorismate synthase" refers to an enzyme that catalyzes the reaction of 5-enolpyruvoylshikimate 3-phosphate to chorismate.

[0019] As used herein, the term "anthranilate synthase" means an enzyme that catalyzes the reaction of chorismate to anthranilate.

[0020] As used herein, the term "ribose-phosphate pyrophosphokinase" refers to an enzyme that catalyzes the reaction of ribose-5-phosphate to phospho-α-D-ribosyl-1-pyrophosphate.

[0021] As used herein, the term "anthranilate phosphoribosyltransferase" refers to an enzyme that catalyzes the reaction of anthranilic acid and phospho-α-D-ribosyl-1-pyrophosphate to N-(5-phosphoribosyl)-anthranilic acid.

[0022] As used herein, the term "N-(5'-phosphoribosyl)anthranilate isomerase" refers to an enzyme that catalyzes the reaction of N-(5-phosphoribosyl)-anthranilic acid to 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate.

[0023] As used herein, the term "indole-3-glycerol phosphate synthase" refers to an enzyme that catalyzes the reaction of 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate to (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate.

[0024] As used herein, the term "tryptophan synthase" refers to an enzyme that catalyzes the conversion of (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate plus serine to L-tryptophan.

[0025] As used herein, the term "tryptophan decarboxylase" refers to an enzyme that catalyzes the reaction of L-tryptophan to tryptamine.

[0026] As used herein, the term "chorismate mutase" means an enzyme that catalyzes the reaction of chorismate to prephenate.

[0027] As used herein, the term "prephenate dehydrogenase" means an enzyme that catalyzes the reaction of prephenate to phenylpyruvate.

[0028] As used herein, the term "aromatic aminotransferase" refers to an enzyme that catalyzes the reaction of phenylpyruvate to phenylalanine.

[0029] As used herein, the term "phenylalanine ammonium lyase" means an enzyme that catalyzes the reaction of phenylalanine to cinnamic acid.

[0030] As used herein, the term "cinnamate 4-hydroxylase" refers to an enzyme that catalyzes the reaction of cinnamic acid to coumaric acid.

[0031] As used herein, the term "CPR" refers to cytochrome P450 reductase, which catalyzes electron transfer (from NADPH) to cytochrome P450, typically in the endoplasmic reticulum of a eukaryotic cell.

[0032] As used herein, the terms "cytochrome P450 enzyme" or "P450 enzyme" or "P450" are used interchangeably and refer to a family of enzymes that contain heme as a cofactor. The P450s of the present invention can catalyze the oxidation, hydroxylation (hydroxylase) of a substrate, and / or the addition of nitrogen dioxide (NO2) to a substrate.

[0033] As used herein, the term "4-Coumaroyl CoA ligase" refers to an enzyme that catalyzes the reaction of coumaric acid to 4-coumaroyl CoA.

[0034] As used herein, the term "tryptophanase" means an enzyme that catalyzes the reaction of tryptophan or a derivative thereof to indole or a derivative thereof.

[0035] As used herein, the term "tryptophan synthase" refers to an enzyme that catalyzes the reaction of indole or a derivative thereof and serine or a derivative thereof to tryptophan or a derivative thereof.

[0036] As used herein, the term "tryptophan decarboxylase" or "nonstandard aromatic amino acid decarboxylase" refers to an enzyme that catalyzes the reaction of tryptophan or a derivative thereof to tryptamine or a derivative thereof.

[0037] The term "glycosyltransferase" or "GT" refers to an enzyme (EC 2.4) that catalyzes the formation of a glycoside by the transfer of a glycosyl group (sugar) from an activated glycosyl donor to a nucleophilic glycosyl acceptor molecule, where the nucleophilic group may be oxygen-, carbon-, nitrogen-, or sulfur-based. The product of glycosyl transfer may be an O-, N-, S-, or C-glycoside. In the present case, the nucleophilic glycosyl acceptor is a tryptamine or tryptophan derivative or a glycosylated tryptamine or tryptophan derivative, and the product of glycosyl transfer is an O- or C-glycoside.

[0038] Glycosyltransferases can be further divided into different GT families according to 3D structure and reaction mechanism. More specifically, the GT1 superfamily refers to UDP glycosyltransferases (UGTs) that contain a PSPG box-linked UDP sugar. UGT superfamily members can be further divided into families and subfamilies according to amino acid identity, as defined by the UGT Nomenclature Committee (Mackenzie et al., 1997).

[0039] As used herein, the terms "N-methyltransferase" and "O-methyltransferase" and "C-methyltransferase" refer to enzymes that catalyze the reactions of methylation of an N, O or C moiety, respectively.

[0040] As used herein, the term "strictosidine synthase" or "1-acetyl-β-carboline synthase" refers to an enzyme that catalyzes the reaction of tryptamine and an aldehyde to a β-carboline.

[0041] As used herein, the term "N-acetyltransferase" refers to an enzyme that catalyzes the reaction of tryptophan or a derivative thereof to N-acetyltryptamine or a derivative thereof.

[0042] As used herein, the term "hydroxytryptamine kinase" refers to an enzyme that catalyzes the reaction of hydroxytryptamine or a derivative thereof to phosphoryloxytryptamine or a derivative thereof. The hydroxytryptamine may be, for example, 4-hydroxytryptamine or 7-hydroxytryptamine.

[0043] As used herein, the term "N-hydroxycinnamoyltransferase" means an enzyme that catalyzes the reaction of tryptamine or a derivative thereof and cinnamoyl CoA to N-cinnamoyltryptamine or a derivative thereof and CoA.

[0044] As used herein, the term "psilocybin phosphatase" refers to an enzyme that catalyzes the reaction of R-phosphoryloxytryptamine or a derivative thereof to R-hydroxytryptamine or a derivative thereof.

[0045] As used herein, the term "psilocin laccase" refers to an enzyme that catalyzes the reaction of R-hydroxytryptamine or a derivative thereof to tryptamine quinoid or a derivative thereof.

[0046] As used herein, the term "tryptophan halogenase" refers to an enzyme that catalyzes the reaction of tryptamine or a derivative thereof to a halogenated tryptamine or a derivative thereof.

[0047] As used herein, the term "lyase" refers to an enzyme that catalyzes the cleavage of bonds that occur during the splitting of a molecule into its separate components.

[0048] As used herein, the term "flavin monooxygenase" refers to an enzyme that catalyzes the oxidation of a substrate using NADPH as a cofactor and flavin adenine dinucleotide (FAD) as a prosthetic group.

[0049] As used herein with respect to glycosyl donors, the term "nucleotide glycoside" refers to a compound that includes a nucleotide moiety covalently linked to a glycosyl group, where the nucleotide includes a nucleoside covalently linked to one or more phosphate groups. Such compounds are also referred to as "activated glycosides," where the glycosyl group is a sugar, such as a "nucleotide sugar" or an "activated sugar."

[0050] As used herein, the terms "heterologous" or "recombinant" and their grammatical equivalents refer to material "from a different species or cell." For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell that does not naturally contain that gene, i.e., the gene is from a different species or cell type than the host cell.

[0051] As used herein, the term "genetically modified host cell" means a host cell that contains and expresses a heterologous or recombinant polynucleotide gene.

[0052] As used herein, the terms "pathway" or "metabolic pathway" or "biosynthetic metabolic pathway" or "operative biosynthetic metabolic pathway" are used interchangeably and are intended to mean one or more enzymes that act in a living cell to convert a chemical substrate into a chemical product. A pathway may include one enzyme or multiple enzymes acting in sequence. A pathway that includes only one enzyme may also be referred to herein as a "bioconversion", especially when the cell is supplied with a precursor or substrate to be converted by the enzyme into a desired product molecule. An enzyme is characterized by having catalytic activity, which can change the chemical structure of a substrate. An enzyme may have more than one substrate and produce more than one product. An enzyme may also depend on a cofactor, which may be an inorganic chemical compound or an organic compound (cofactor and / or coenzyme). NADPH-dependent cytochrome P450 reductase (CPR) is the electron donor for cytochrome P450 (CYP). CPR shuttles electrons from NADPH through flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) coenzymes to the iron of the substituted heme group of CYP.

[0053] As used herein, the term "in vivo" means within a living cell or within an organism, including, for example, an animal, plant, or microorganism.

[0054] As used herein, the term "in vitro" means outside a living cell or organism, including, but not limited to, for example, a microwell plate, a tube, a flask, a beaker, a tank, a reactor, and the like.

[0055] As used herein, the term "substrate" or "precursor" refers to any compound that can be converted into a different compound. For example, thebaine is a substrate for P450 and can be converted to northebaine by demethylation. For clarity, substrate and / or precursor include both compounds generated in situ by enzymatic reactions in exogenously provided compounds, such as organic molecules that a cell or host cell can metabolize into a compound of interest.

[0056] As used herein, the term "endogenous" or "wild type" refers to a gene or polypeptide in a host cell that originates from the same host cell.

[0057] As used herein, the term "deletion" refers to the manipulation of a gene such that it is no longer expressed in the host cell.

[0058] As used herein, the term "disruption" refers to the manipulation of a gene or any machinery involved in expression such that it is no longer expressed in the host cell.

[0059] As used herein, the term "attenuating" refers to the manipulation of a gene or any mechanism involved in the expression of the gene such that expression of the gene is reduced compared to expression in the absence of the manipulation.

[0060] As used herein, the term "substantially" or "approximately" or "about" refers to a reasonable deviation in the vicinity of a value or parameter such that the value or parameter does not change significantly. These deviation terms from a value should be interpreted as including deviations in the value when the deviation does not negate the meaning of the value from which it deviates. For example, in connection with a reference numerical value, a term of degree can include a range of values ​​of ±10% from that value. For example, the use of these deviation terms can include a ± range of deviation, for example, ±9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0061] As used herein, the term "and / or" is intended to represent the inclusive "or." The terms X and / or Y are intended to mean either X or Y, as well as both X and Y. Furthermore, the terms X, Y and / or Z are intended to mean either X, Y and Z alone or any combination of X, Y and Z.

[0062] As used herein, the terms "isolated" or "produced" or "extracted" or "recovered" are used interchangeably with respect to a compound and refer to any compound that has been placed, by human intervention, into a form or environment different from that in which it is found in nature. An isolated compound includes, but is not limited to, a compound of the invention in which the ratio of the compound to other components with which it naturally accompanies is increased or decreased. In important embodiments, the amount of the compound is increased relative to other components with which it naturally accompanies. In certain embodiments, the compounds of the invention may be isolated in a pure or substantially pure form. In this context, a substantially pure compound means that the compound is separated from other extraneous or undesirable substances that are present from the start of the production of the compound or that are produced during the manufacturing process. Such a substantially pure compound preparation contains less than 10% by weight, such as less than 8% by weight, such as less than 6% by weight, such as less than 5% by weight, such as less than 4% by weight, such as less than 3% by weight, such as less than 2% by weight, such as less than 1% by weight, such as less than 0.5% by weight of other extraneous or undesirable substances that normally accompany the compound when naturally or recombinantly expressed. In an embodiment the isolated compound is at least 90% by weight pure, such as at least 91% by weight pure, such as at least 92% by weight pure, for example at least 93% by weight pure, such as at least 94% by weight pure, for example at least 95% by weight pure, such as at least 96% by weight pure, for example at least 97% by weight pure, such as at least 98% by weight pure, for example at least 99% by weight pure, such as at least 99.5% by weight pure, for example 100% by weight pure.

[0063] The term "% identity" is used herein for the relatedness between two amino acid sequences or between two nucleotide sequences. When used herein for amino acid sequences, "% identity" refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably the Needle program 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

number

number

[0064] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing.

[0065] The term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are usually determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0066] As used herein, the term "control sequence" refers to a nucleotide sequence necessary for the expression of a polynucleotide encoding a polypeptide. The control sequence may be native (from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide coding sequences, promoter sequences, signal peptide coding sequences, translation termination (stop) sequences, and transcription termination (stop) sequences. To be operable, control sequences must usually include promoter sequences, transcriptional and translational stop signals. Control sequences may be provided with linkers to introduce specific restriction enzyme sites facilitating ligation of the control sequence with the coding region of the polynucleotide encoding the polypeptide.

[0067] The term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0068] The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to a control sequence that provides for its expression.

[0069] The term "host cell" refers to any cell type that can be transformed, transduced, transduced, etc., with a polynucleotide construct or expression vector containing a polynucleotide of the invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0070] The term "polynucleotide construct" means a single- or double-stranded polynucleotide that is isolated from a naturally occurring gene or that has been modified or synthesized to contain a segment of nucleic acid that would not otherwise be present in nature and that contains one or more regulatory sequences.

[0071] The term "operably linked" refers to a configuration in which a control sequence is positioned in an appropriate position relative to a coding polynucleotide such that the control sequence directs the expression of the coding polynucleotide.

[0072] The terms "nucleotide sequence" and "polynucleotide" are used interchangeably herein.

[0073] The terms "comprise" and "include" as well as conjugations such as "comprises," "comprising," "includes," and "including" used throughout the specification and the appended claims are to be construed as inclusive. These phrases are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0074] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., one or at least one) of the grammatical object of the article. For example, "an element" can mean one element or two or more than one elements.

[0075] Terms such as "preferably," "generally," "particularly," and "typically" are not used herein to limit the scope of the claimed invention or to imply that particular features are critical, essential, or critical to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be available in a particular embodiment of the invention.

[0076] As used herein, the term "cell culture" refers to a medium containing a plurality of genetically modified host cells of the present invention. The cell culture may contain a single strain of genetically modified host cells, or may contain two or more separate strains of genetically modified host cells. The medium may be any medium suitable for the genetically modified host cells, such as a liquid medium (i.e., culture medium) or a semi-solid medium, and may contain additional components, such as a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleic acid bases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B.

[0077] Method for producing tryptamine derivatives The present invention relates to a compound of formula (I): [ka] wherein tryptamine derivative (I) is not tryptophan, 4-hydroxytryptamine, N-acetyl-4-hydroxytryptamine, norbaeocystin, baeocystin; psilocybin, psilocin, aeruginasin, halogenated tryptophan, halogenated tryptamine, halogenated N-methylated tryptamine, halogenated N,N-dimethyltryptamine or halogenated N,N,N-trimethyltryptamine; said method comprises producing a tryptamine derivative of formula (II): [ka] providing an indole acceptor of formula R II ~R VII is not H, and R III is H or CH2CH2NH2 or CH2CHCOOHNH2; and further, the method includes contacting the indole acceptor with a substituent donor in the presence of one or more enzymes that replace one or more of the H, OH and / or COOH in the indole acceptor with one or more substituents of the substituent donor.

[0078] In some embodiments, the R of the indole receptor (II) II , R IV , R V , R VI , and / or R VII is OH, Cl, Br, F, I, CH3, NO2, PO4, or CH3-O. Particularly useful are tryptamine derivatives in which R4 and / or R5 of formula I are OH. More specifically, the indole receptor (II) is selected from 4-hydroxytryptamine, 5-hydroxytryptamine, psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, aeruginasin, 4-hydroxy-N,N,N-trimethyltryptamine, bufotenin, norbufotenin, 5-hydroxy-N,N,N-trimethyltryptamine, 4-methoxytryptamine, 5-methoxytryptamine, N-acetylserotonin, ibogaine, ibogamine, noribogaine, mitragynine, 7-OH-mitragynine, 4-HO-DET, 4-HO-DiPT, 4-HO-MET, 4-HO-MiPT, 4-HO-McPT, 4-HO-DPT, 4-HO-DSBT, and / or harmalol, which indole receptors are further derivatized with at least one additional substitution that provides the molecule with improved properties.

[0079] In some embodiments, the substituent transferred to the indole acceptor is an alkyl group, an acetyl group, a glycosyl group, a phosphate group, an oxygenyl group, a hydroxyl group, or a halogenyl group. When the substitutent is a glycosyl group, the glycosyl moiety of the glycosyl group preferably comprises one or more sugars selected from glucose, galactose, xylose, mannose, galactofuranose, arabinose, rhamnose, apiose, fucose, glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine, xylosamine, mannosamine, arabinosamine, rhamnosamine, apiosamine, fucosamine, glucuronic acid, galacturonic acid, mannuronic acid, arabic acid, apionic acid, or combinations thereof. Substitution with a glycosyl group may be suitable for O-glycosylation, such as β-O-glycosylation.

[0080] When the substituent is an alkyl group, the alkyl group is suitably an ethyl or methyl group. The alkylation reaction is suitably O-alkylation, N-alkylation or C-alkylation, optionally ethylation or methylation.

[0081] When the substituent is an acetyl group, the acetylation reaction is suitably N-acetylation. An example of an acetyl donor is acetyl-CoA.

[0082] The donors that provide the substituents to the indole acceptor can be aldehydes, ketones, ethers and / or amines.

[0083] Suitable aldehydes include acetaldehyde, oxaloacetaldehyde and / or secologanin, while suitable ketones include cinnamoyl CoA or pyruvate.

[0084] The ether may be a glycoside, particularly a nucleotide glycoside, such as an NTP-glycoside, an NDP-glycoside, or an NMP-glycoside. In some embodiments, the nucleoside of the nucleotide glycoside is selected from uridine, adenosine, guanosine, cytidine, and / or deoxythymidine. In particular, the nucleotide glycoside includes a UDP-glycoside, an ADP-glycoside, a CDP-glycoside, a CMP-glycoside, a dTDP-glycoside, and / or a GDP-glycoside. Particularly useful nucleotide glycosides are UDP-D-glucose (UDP-Glc); UDP-galactose (UDP-Gal); UDP-D-xylose (UDP-Xyl); UDP-N-acetyl-D-glucosamine (UDP-GlcNAc); UDP-N-acetyl-D-galactosamine (UDP-GalNAc); UDP-D-glucuronic acid (UDP-GlcA); UDP-D-galactofuranose (UDP-Galf); UDP-arabinose; UDP-rhamnose, UDP-apiose; UDP-2-acetamido-2-deoxy-α-D-mannuronic acid; UDP-N-acetyl-D-galactosamine. 4-sulfate; UDP-N-acetyl-D-mannosamine; UDP-2,3-bis(3-hydroxytetra-decanoyl)glucosamine; UDP-4-deoxy-4-formamido-β-L-arabinopyranose; UDP-2,4-bis(acetamido)-2,4,6-trideoxy-α-D-glucopyranose; UDP-galacturonic acid; UDP-3-amino-3-deoxy-α-D-glucose; guanosine diphospho-D-mannose (GDP-Man); guanosine diphospho-L-fucose (GDP-Fuc); guanosine diphospho-L-rhamnose (GDP-Rha); cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac); cytidine monophospho-2-keto-3-deoxy-D-mannooctanoic acid (CMP-Kdo); and ADP-glucose.

[0085] The amine can be S-adenosylmethionine (SAM) or S-adenosylethionine (SAE).

[0086] In the methods of the invention, the one or more enzymes are suitably glycosyltransferases, alkyltransferases, synthases, acetyltransferases, kinases, cinnamoyltransferases, phosphatases, laccases, halogenases, P450 enzymes, flavin monooxygenases, and / or lyases.

[0087] The glycosyltransferase of the present invention may be derived from a plant, such as rice (Oryza sativa), saffron (Crocus sativus), tobacco (Nicotiana tabacum), stevia (Stevia rebaudiana), tobacco (Nicotiana benthamiana) and / or Arabidopsis thaliana, or from a fungus. The glycosyltransferase may be an O-glycoside transferase that transfers a glycosyl group to the O of an indole acceptor and / or a C-glycoside transferase that transfers a glycosyl group to the C of an indole acceptor. In some embodiments, the glycosyltransferase can O-glycosylate an aglycone acceptor or a glycosylated acceptor or both. In either case, the glycosyl group may be glucose, rhamnose, xylose, arabinose, N-acetylgalactosamine, or N-acetylglucosamine. In further embodiments, the glycosyltransferase can transfer a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide to an indole acceptor, such as an aglycone / glycoside mono-O-glycosyltransferase, a di-O-glycosyltransferase, a tri-O-glycosyltransferase, or a tetra-O-glycosyltransferase, respectively. In a preferred embodiment, the glycosyltransferase is a hydroxytryptamine glycosyltransferase. In alternative embodiments, the glycosyltransferase of the present invention can be selected from the EC classes EC2.4.1.-, and / or EC2.4.2.-, such as EC2.4.1.17, EC2.4.1.35, EC2.4.1.159, EC2.4.1.203, EC2.4.1.234, EC2.4.1.236, EC2.4.1.294, and / or EC2.4.2.40.Examples of suitable glycosyltransferases are those included in SEQ ID NOs: 80, 82, 84, or 86, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250 and / or 252. The glycosyltransferase of the present invention preferably has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a glycosyltransferase included in any of SEQ ID NOs: 80, 82, 84, or 86, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250 and / or 252.

[0088] In some embodiments, the glycosyltransferase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a glycosyltransferase included in any of SEQ ID NOs: 194 and / or 204.

[0089] The alkyltransferase of the present invention can be a methyltransferase, such as an O-methyltransferase, an N-methyltransferase, or a C-methyltransferase. Suitable O-methyltransferases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to an O-methyltransferase included in any of SEQ ID NOs: 114, 116, 118, and / or 120. Suitable N-methyltransferases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to an N-methyltransferase included in any of SEQ ID NOs: 122, 124, 126, 128, 130, 132, 134, 136, and / or 138. Suitable C-methyltransferases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a C-methyltransferase included in SEQ ID NO: 140.

[0090] The synthase of the present invention can be a strictosidine synthase or a 1-acetyl-β-carboline synthase. Suitable strictosidine synthases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a strictosidine synthase included in any of SEQ ID NOs: 144, 146, 148 and / or 150. Suitable 1-acetyl-β-carboline synthases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a 1-acetyl-β-carboline synthase included in any of SEQ ID NOs: 152 and / or 154.

[0091] The acetyltransferase may be an aralkylamine N-acetyltransferase, including those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to an aralkylamine N-acetyltransferase contained in SEQ ID NO: 142.

[0092] Kinases of the invention may be 4-hydroxytryptamine kinases and / or 7-hydroxytryptamine kinases including those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a 4-hydroxytryptamine kinase and / or a 7-hydroxytryptamine kinase included in any of SEQ ID NOs: 156, 158, and / or 160.

[0093] The cinnamoyltransferase of the present invention may be an N-hydroxycinnamoyltransferase including those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the N-hydroxycinnamoyltransferase contained in SEQ ID NO: 162.

[0094] The phosphatase of the present invention may be a psilocybin phosphatase including those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the psilocybin phosphatase contained in SEQ ID NO: 164.

[0095] The laccase of the present invention may be a psilocin laccase including one having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the psilocin laccase contained in SEQ ID NO: 166.

[0096] The halogenase may be a tryptophan halogenase, such as tryptophan 2-halogenase, tryptophan 5-halogenase, tryptophan 6-halogenase, or tryptophan 7-halogenase. Suitable tryptophan 2-halogenases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the tryptophan 2-halogenase contained in SEQ ID NO: 168. Suitable tryptophan 5-halogenases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the tryptophan 5-halogenase contained in SEQ ID NO: 170. Suitable tryptophan 6-halogenases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan 6-halogenase contained in SEQ ID NO: 172. Suitable tryptophan 7-halogenases include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan 7-halogenase contained in SEQ ID NO: 174.

[0097] P450 enzymes of the present invention include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a P450 enzyme included in any of SEQ ID NOs: 88, 90, 92, 94, 96, 100 and / or 178, and the method optionally further comprises contacting the P450 with a P450 reductase (CPR). In certain embodiments, the P450 enzyme has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the P450 enzyme contained in SEQ ID NO: 96 (OsT5H), and optionally the P450 reductase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the P450 reductase contained in SEQ ID NO: 112 (FoCPR).

[0098] Flavin monooxygenases of the present invention include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a flavin monooxygenase included in SEQ ID NO:98.

[0099] Lyases of the present invention include those having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a lyase included in any of SEQ ID NOs: 52 or 176.

[0100] The sequence identity to said replacement enzyme may be at least 90%, such as at least 95%, such as at least 99%, such as 100%, such as especially at least 99%, such as even 100%.

[0101] The tryptamine derivative (I) of the present invention may be a hydroxytryptamine β-O-glycoside, such as a hydroxytryptamine β-O-glycoside, including, but not limited to, 4-hydroxytryptamine-β-O-glycoside, psilocin-β-O-glycoside, norpsilocin-β-O-glycoside, 4-hydroxy-N,N,N-trimethyltryptamine-β-O-glycoside, serotonin-β-O-glycoside, bufotenin-β-O-glycoside, norbufotenin-β-O-glycoside, 5-hydroxy-N,N,N-trimethyltryptamine-β-O-glycoside, N-acetylserotonin-β-O-glycoside, ... and / or 4-HO-aminobutyric acid, ...

[0102] In a further embodiment, the method of the invention comprises one or more steps selected from: a) converting indole or an indole derivative to tryptophan or a tryptophan derivative; and b) converting tryptophan or a tryptophan derivative to tryptamine or a tryptamine derivative.

[0103] These steps a) and / or b) may be carried out in vitro.

[0104] In particular, conversion of indole or an indole derivative to tryptophan or a tryptophan derivative may comprise contacting the indole or an indole derivative with a tryptophan synthase enzyme, such as a tryptophan synthase having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan synthase contained in SEQ ID NO: 60, 62, 64, 66, 68, 180, 182 and / or 256. In certain embodiments, the tryptophan synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan synthase contained in SEQ ID NO: 256.

[0105] Further, conversion of tryptophan or a tryptophan derivative to tryptamine or a tryptamine derivative can include contacting the tryptophan or tryptophan derivative with a tryptophan decarboxylase enzyme, such as a tryptophan synthase having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan decarboxylase enzyme, e.g., a tryptophan synthase included in SEQ ID NOs: 26, 70, 72, 74, 76 and / or 78. In certain embodiments, conversion of indole or an indole derivative to tryptophan or a tryptophan derivative comprises contacting the indole or an indole derivative with a tryptophan synthase enzyme having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, for example at least 95%, such as at least 99%, for example 100% identity to a tryptophan synthase contained in SEQ ID NOs: 180 and / or 256 in the presence of a tryptophan decarboxylase having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, for example at least 95%, such as at least 99%, for example 100% identity to a tryptophan decarboxylase contained in SEQ ID NOs: 72 and / or 78.

[0106] In an important embodiment, the indole receptor is serotonin, the tryptamine derivative (I) is serotonin, optionally a derivative of melatonin, normelatonin, or a hydroxycinnamoyl serotonin such as 4-coumaroyl serotonin, and the one or more enzymes that replace one or more of the H, OH and / or COOH in the indole receptor with one or more substituents of a substituent donor are selected from: a) an acetyltransferase having at least 70% identity to the acetyltransferase contained in SEQ ID NO: 142; b) an O-methyltransferase having at least 70% identity to the O-methyltransferase contained in SEQ ID NO: 118; and / or c) An N-hydroxycinnamoyltransferase having at least 70% identity to Cin trans contained in SEQ ID NO: 162.

[0107] The method of the invention may further comprise one or more additional steps selected from: a) glycosylation; b) methylation; c) hydroxylation; d) condensation; e) nitration; f) oxidation; g) lyase deamidation; or h) Dephosphorylation.

[0108] The hydroxylation step may optionally comprise contacting the indole or indole derivative or tryptophan or tryptophan derivative with a hydroxylase, including one having at least 70%, such as at least 75%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 99%, for example 100% identity to a cytochrome P450 reductase (CPR) comprised in any of SEQ ID NOs: 102, 104, 106, 108, 110 and / or 112, in the presence of a CRP having at least 70%, such as at least 75%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 99%, for example 100% identity to a hydroxylase comprised in any of SEQ ID NOs: 88, 90, 92, 94, 96, 98 and / or 100.

[0109] The lyase deamidation step may comprise contacting the indole or indole derivative or tryptophan or tryptophan derivative with a lyase including one having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a lyase included in any of SEQ ID NOs: 52 or 176.

[0110] In a preferred embodiment, the method includes an in vitro enzyme replacement step and / or, optionally, an in vivo enzyme replacement step. In particular, the method may include expressing a glycosyltransferase in E. coli and performing in vitro glycosylation of an indole acceptor.

[0111] Tryptamine Derivatives The present invention also provides novel tryptamine derivatives obtainable from the process of the present invention. Such tryptamine derivatives have the formula (I): [ka] wherein at least one of R2, R4 to R9, α and β is a glycosyl group, and in particular R2, R4 to R7, α and β can be an O-glycosyl group. Such tryptamine derivatives include, but are not limited to, 4-hydroxytryptamine-β-O-glycoside, psilocin-β-O-glycoside, norpsilocin-β-O-glycoside, 4-hydroxy-N,N,N-trimethyltryptamine-β-O-glycoside, serotonin-β-O-glycoside, bufotenin-β-O-glycoside, norbufotenin-β-O-glycoside or 5-hydroxy-N,N,N-trimethyltryptamine-β-O-glycoside, N-acetylserotonin-β-O-glycoside, noribogaine-β-O-glycoside, 7-OH-mitragynyl These glycosides include 4-HO-glycan-β-O-glycoside, 4-HO-DET-β-O-glycoside, 4-HO-DiPT-β-O-glycoside, 4-HO-MET-β-O-glycoside, 4-HO-MiPT-β-O-glycoside, 4-HO-McPT-β-O-glycoside, 4-HO-DPT-β-O-glycoside, and 4-HO-DSBT-β-O-glycoside, while the glycosides of serotonin-β-O-glycoside, bufotenin-β-O-glycoside, norbufotenin-β-O-glycoside, or 5-hydroxy-N,N,N-trimethyltryptamine-β-O-glycoside are not glycosides.

[0112] Genetically modified host cells and cell culture In a further aspect of the present invention, as described above, the method is carried out in a host cell genetically modified to produce the tryptamine derivative of the present invention.Therefore, the present invention also provides a host cell genetically modified to produce tryptamine derivative in the presence of a substituent donor, the host cell expresses one or more heterologous genes encoding one or more replacement enzymes, which in the presence of a substituent donor and an indole acceptor, transfer the substituent from the donor to the acceptor, thereby producing a substituted tryptamine.The replacement enzyme and donor for carrying out the method in a genetically modified cell are suitable as described above for the method.

[0113] The genetically modified microbial host cell suitably expresses one or more genes selected from the following: a) a gene encoding a UGT, or a genomic DNA thereof, that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a UGT-encoding polynucleotide comprised in any of SEQ ID NOs: 79, 81, 83, 85, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and / or 251; b) a gene encoding an O-methyltransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to an O-methyltransferase-encoding polynucleotide comprised in any of SEQ ID NOs: 113, 115, 117 and / or 119, or a genomic DNA thereof; c) a gene encoding an N-methyltransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to an N-methyltransferase-encoding polynucleotide comprised in any of SEQ ID NOs: 121, 123, 125, 127, 129, 131, 133, 135, and / or 137, or a genomic DNA thereof; d) a gene encoding a C-methyltransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the C-methyltransferase-encoding polynucleotide contained in SEQ ID NO: 139, or its genomic DNA; e) a gene encoding a strictosidine synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a strictosidine synthase-encoding polynucleotide comprised in any of SEQ ID NOs: 143, 145, 147, and / or 149, or a genomic DNA thereof; f) a gene encoding a 1-acetyl-β-carboline synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a 1-acetyl-β-carboline synthase-encoding polynucleotide included in any of SEQ ID NOs: 151 and / or 153, or a genomic DNA thereof; g) a gene encoding a 1-acetyl-β-carboline synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to an aralkylamine N-acetyltransferase-encoding polynucleotide contained in SEQ ID NO: 141, or a genomic DNA thereof; h) a gene encoding 4-hydroxytryptamine kinase and / or 7-hydroxytryptamine kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a 4-hydroxytryptamine kinase and / or 7-hydroxytryptamine kinase encoding polynucleotide contained in any of SEQ ID NOs: 155, 157, and / or 159, or a genomic DNA thereof; i) a gene encoding an N-hydroxycinnamoyltransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the N-hydroxycinnamoyltransferase-encoding polynucleotide contained in SEQ ID NO: 161, or a genomic DNA thereof; j) a gene encoding a psilocybin phosphatase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the psilocybin phosphatase-encoding polynucleotide contained in SEQ ID NO: 163, or a genomic DNA thereof; k) a gene encoding a psilocin laccase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the psilocin laccase-encoding polynucleotide contained in SEQ ID NO: 165, or a genomic DNA thereof; l) a gene encoding a tryptophan 2-halogenase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the tryptophan 2-halogenase-encoding polynucleotide contained in SEQ ID NO: 167, or a genomic DNA thereof; m) a gene encoding a tryptophan 5-halogenase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the tryptophan 5-halogenase-encoding polynucleotide contained in SEQ ID NO: 169, or a genomic DNA thereof; n) a gene encoding a tryptophan 6-halogenase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the tryptophan 6-halogenase-encoding polynucleotide contained in SEQ ID NO: 171, or a genomic DNA thereof; o) a gene encoding a tryptophan 7-halogenase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the tryptophan 7-halogenase-encoding polynucleotide contained in SEQ ID NO: 173, or a genomic DNA thereof; p) a gene encoding a P450 enzyme that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a P450 enzyme encoding polynucleotide comprised in any of SEQ ID NOs: 87, 89, 91, 93, 95, 99, and / or 177, or a genomic DNA thereof; q) a gene encoding a P450 reductase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a P450 reductase (CPR) encoding polynucleotide comprised in any of SEQ ID NOs: 101, 103, 105, 107, 109, and / or 111, or a genomic DNA thereof; r) a gene encoding a flavin monooxygenase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a flavin monooxygenase-encoding polynucleotide contained in SEQ ID NO: 97, or a genomic DNA thereof; s) a gene encoding a lyase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a lyase-encoding polynucleotide comprised in any of SEQ ID NOs: 51, and / or 175, or a genomic DNA thereof.

[0114] The host cells of the invention may further comprise an operative biosynthetic pathway to produce an indole acceptor, wherein the host cell expresses a polypeptide encoding one or more pathway genes selected from: a) one or more enzymes that convert glucose to fructose-6-phosphate; b) fructose-6-phosphate phosphoketolase, which converts fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate; c) phosphotransacetylase, which converts acetyl phosphate to acetyl CoA; d) one or more enzymes that convert fructose-6-phosphate to phosphoenolpyruvate; e) 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase), which converts phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP); f) one or more enzymes that convert 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimate 3-phosphate; g) shikimate kinase, which converts shikimate to shikimate-3-phosphate; h) chorismate synthase, which converts 5-enolpyruvoylshikimate 3-phosphate to chorismate; i) anthranilate synthase, which converts chorismate to anthranilate; j) ribose-phosphate pyrophosphokinase, which converts ribose-5-phosphate to phospho-α-D-ribosyl-1-pyrophosphate; k) anthranilate phosphoribosyltransferase, which converts anthranilate and phospho-α-D-ribosyl-1-pyrophosphate to N-(5-phosphoribosyl)-anthranilic acid; l) N-(5'-phosphoribosyl)anthranilate isomerase, which converts N-(5-phosphoribosyl)-anthranilate to 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate; m) indole-3-glycerol phosphate synthase, which converts 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate to (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate; n) tryptophan synthase, which converts (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate and serine to L-tryptophan; o) tryptophan decarboxylase, which converts L-tryptophan to tryptamine; p) chorismate mutase, which converts chorismate to prephenate; q) prephenate dehydrogenase, which converts prephenate to phenylpyruvate; r) aromatic aminotransferase, which converts phenylpyruvate to phenylalanine; s) phenylalanine ammonium lyase, which converts phenylalanine to cinnamic acid; t) cinnamate 4-hydroxylase, which converts cinnamic acid to coumaric acid; u) cytochrome b5, which assists cytochrome P450 reductase in reducing hydroxylase enzymes; v) cytochrome P450 reductase, which reduces cytochrome P450 enzymes; w) 4-coumaroyl-CoA ligase, which converts coumaric acid to 4-coumaroyl-CoA; x) tryptophanase, which converts tryptophan or its derivatives into indole or its derivatives; y) tryptophan synthase, which converts indole or its derivatives and serine or its derivatives into tryptophan or its derivatives; z) tryptophan decarboxylase or nonstandard aromatic amino acid decarboxylase that converts tryptophan or its derivatives to tryptamine or its derivatives; aa) tryptamine 5-hydroxylase, which converts tryptamine to serotonin; bb) tryptamine 4-hydroxylase, which converts tryptamine to 4-hydroxytryptamine; cc) 4-hydroxytryptamine kinase, which converts 4-hydroxytryptamine to norbeocystin; and / or dd) psilocybin synthase, which converts norbeocystin to psilocybin.

[0115] In some embodiments of the host cell, a) the fructose-6-phosphate phosphoketolase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the fructose-6-phosphate phosphoketolase contained in SEQ ID NO:2; b) the phosphotransacetylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the phosphotransacetylase contained in SEQ ID NO:4; c) the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase contained in SEQ ID NO:6; d) an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimate 3-phosphate has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the enzyme contained in SEQ ID NO:8; e) the shikimate kinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the shikimate kinase contained in SEQ ID NO: 10; f) the chorismate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the chorismate synthase contained in SEQ ID NO: 12; g) the anthranilate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the anthranilate synthase contained in SEQ ID NO: 14; h) the ribose-phosphate pyrophosphokinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ribose-phosphate pyrophosphokinase contained in SEQ ID NO: 16; i) the anthranilate phosphoribosyltransferase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the anthranilate phosphoribosyltransferase contained in SEQ ID NO: 18; j) the N-(5'-phosphoribosyl)anthranilate isomerase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the N-(5'-phosphoribosyl)anthranilate isomerase contained in SEQ ID NO: 20; k) the indole-3-glycerol synthase phosphate has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the indole-3-glycerol synthase phosphate contained in SEQ ID NO: 22; l) the tryptophan synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan synthase in any of SEQ ID NOs: 24, 60, 62, 64, 66, 68, 180 and / or 182; m) the tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase included in any of SEQ ID NOs: 26, 70, 72, 74, 76, and / or 78; n) the chorismate mutase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the chorismate mutase contained in SEQ ID NO: 46; o) the prephenate dehydrogenase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the prephenate dehydrogenase contained in SEQ ID NO: 48; p) the aromatic aminotransferase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to an aromatic aminotransferase contained in SEQ ID NO: 50; q) the phenylalanine ammonium lyase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the phenylalanine ammonium lyase contained in SEQ ID NO: 52; r) the cinnamate 4-hydroxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the cinnamate 4-hydroxylase contained in SEQ ID NO: 54; s) the cytochrome b5 has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the cytochrome b5 contained in SEQ ID NO: 254; t) the cytochrome P450 reductase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a cytochrome P450 reductase included in any of SEQ ID NOs: 56, 102, 104, 106, 108, 110 and / or 112; u) the 4-coumaroyl-CoA ligase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the 4-coumaroyl-CoA ligase contained in SEQ ID NO: 58; v) the tryptamine 5-hydroxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the 5-hydroxylase contained in SEQ ID NO: 96; w) the tryptamine 4-hydroxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the tryptamine 4-hydroxylase contained in SEQ ID NO: 94; x) the 4-hydroxytryptamine kinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the 4-hydroxytryptamine kinase contained in SEQ ID NO: 160; and / or y) the psilocybin synthase has at least 70%, such as at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity to the psilocybin synthase contained in SEQ ID NO: 128.

[0116] In other embodiments of the host cell, the one or more expressed genes are selected from the following: a) a gene encoding a fructose-6-phosphate phosphoketolase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the fructose-6-phosphate phosphoketolase-encoding polynucleotide contained in SEQ ID NO:1, or a genomic DNA thereof; b) a gene encoding a phosphotransacetylase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the phosphotransacetylase-encoding polynucleotide contained in SEQ ID NO:3, or its genomic DNA; c) a gene encoding 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase-encoding polynucleotide contained in SEQ ID NO:5, or a genomic DNA thereof; d) a polynucleotide encoding an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimic acid 3-phosphate and a gene encoding an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimic acid 3-phosphate that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical, or a genomic DNA thereof; e) a gene encoding a shikimate kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a shikimate kinase-encoding polynucleotide contained in SEQ ID NO: 9, or a genomic DNA thereof; f) a gene encoding shikimate kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the chorismate synthase encoding polynucleotide contained in SEQ ID NO: 11, or its genomic DNA; g) a gene encoding an anthranilate synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the anthranilate synthase-encoding polynucleotide contained in SEQ ID NO: 13, or a genomic DNA thereof; h) a gene encoding a ribose-phosphate pyrophosphokinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the ribose-phosphate pyrophosphokinase-encoding polynucleotide contained in SEQ ID NO: 15, or a genomic DNA thereof; i) a gene encoding an anthranilate phosphoribosyltransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the anthranilate phosphoribosyltransferase-encoding polynucleotide contained in SEQ ID NO: 17, or a genomic DNA thereof; j) a gene encoding an N-(5'-phosphoribosyl)anthranilic acid isomerase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the N-(5'-phosphoribosyl)anthranilic acid isomerase-encoding polynucleotide contained in SEQ ID NO: 19, or a genomic DNA thereof; k) a gene encoding an indole-3-glycerol synthase phosphate that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the indole-3-glycerol synthase phosphate-encoding polynucleotide contained in SEQ ID NO: 21, or a genomic DNA thereof; l) a gene encoding a tryptophan synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptophan synthase-encoding polynucleotide comprised in any of SEQ ID NOs: 23, 59, 61, 63, 65, 67, 179, and / or 181, or a genomic DNA thereof; m) tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase encoding genes or genomic DNA thereof that are at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase encoding polynucleotide included in any of SEQ ID NOs: 25, 69, 71, 73, 75, and / or 77; n) a gene encoding a chorismate mutase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the chorismate mutase-encoding polynucleotide contained in SEQ ID NO: 45, or a genomic DNA thereof; o) a gene encoding a prephenate dehydrogenase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the prephenate dehydrogenase-encoding polynucleotide contained in SEQ ID NO: 47, or a genomic DNA thereof; p) a gene encoding an aromatic aminotransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to an aromatic aminotransferase-encoding polynucleotide contained in SEQ ID NO: 49, or a genomic DNA thereof; q) a gene encoding a phenylalanine ammonium lyase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the phenylalanine ammonium lyase-encoding polynucleotide contained in SEQ ID NO: 51, or a genomic DNA thereof; r) a gene encoding a cinnamic acid 4-hydroxylase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the cinnamic acid 4-hydroxylase-encoding polynucleotide contained in SEQ ID NO: 53, or a genomic DNA thereof; s) a gene encoding a cytochrome b5 that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the cytochrome b5-encoding polynucleotide contained in SEQ ID NO: 253, or a genomic DNA thereof; t) a gene encoding a cytochrome P450 reductase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a cytochrome P450 reductase-encoding polynucleotide included in any of SEQ ID NOs: 55, 101, 103, 105, 107, 109, and / or 111, or a genomic DNA thereof; u) a gene encoding a 4-coumaroyl-CoA ligase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the 4-coumaroyl-CoA ligase-encoding polynucleotide contained in SEQ ID NO: 57, or a genomic DNA thereof; v) a gene encoding a tryptamine 5-hydroxylase that is at least 70% identical to the tryptamine 5-hydroxylase-encoding polynucleotide contained in SEQ ID NO: 96, or the genomic DNA thereof; w) a gene encoding a cytochrome P450 reductase that is at least 70% identical to the cytochrome P450 reductase-encoding polynucleotide contained in SEQ ID NO: 111, or the genomic DNA thereof; x) a gene encoding a 4-hydroxytryptamine kinase that is at least 70% identical to the 4-hydroxytryptamine kinase-encoding polynucleotide contained in SEQ ID NO: 159; and / or y) A gene encoding a psilocybin synthase that is at least 70% identical to the psilocybin synthase-encoding polynucleotide contained in SEQ ID NO:127.

[0117] In a preferred embodiment, the host cell of the invention further expresses: a) a gene encoding a psilocybin synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a psilocybin synthase-encoding polynucleotide comprised in any of SEQ ID NOs: 127 and / or 123, or a genomic DNA thereof; b) a gene encoding a 4-hydroxytryptamine kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the 4-hydroxytryptamine kinase-encoding polynucleotide contained in SEQ ID NO: 159, or a genomic DNA thereof; c) a gene encoding a P450 reductase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a P450 reductase (CPR) encoding polynucleotide included in any of SEQ ID NOs: 105 and / or 101, or a genomic DNA thereof; d) a gene encoding a P450 enzyme that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a P450 enzyme-encoding polynucleotide included in any of SEQ ID NOs: 93 and / or 87, or a genomic DNA thereof; and e) a gene encoding a tryptophan decarboxylase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptophan decarboxylase-encoding polynucleotide comprised in any of SEQ ID NOs: 77 and / or 71, or a genomic DNA thereof.

[0118] The host cell comprises at least two copies of one or more heterologous genes encoding replacement or pathway enzymes, and such genes may also be overexpressed. The host cell may also be modified to provide increased amounts of substrate for at least one enzyme of the indole receptor pathway. The host cell may also be further genetically modified to exhibit enhanced tolerance to one or more substrate, intermediate, or product molecules from the indole receptor pathway.

[0119] The host cell of the present invention is suitable to be a eukaryotic, prokaryotic or archaeal cell. The eukaryotic cell is preferably mammalian, insect, plant or fungal. Fungal host cells include those from Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Chytridiomycota, Blastocladiomycota, Zygomycota, Oomycota and Microsporidia. The fungal host cell may be a yeast selected from ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and Fungi lmperfecti yeast (Blastomyces). More specifically, the yeast host cell is from the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces. More specifically, yeast host cells include Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces lactis, Saccharomyces cerevisiae ...The fungal host cells amy are also filamentous fungi, including those from the phyla Ascomycota, Eumycota and Oomycota. More specifically, the filamentous fungal host cell may be selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Corio / us, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, and the like. The fungus may be from the genera: Azotobacter, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma. More specifically, the filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillusniger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporiuminops, Chrysosporium keratinophilum keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium chrookurwelens crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viridae.Alternatively, the host cell is a prokaryotic cell, such as E. coli, or an archaeal cell, such as an algae.

[0120] In important embodiments, one or more host wild-type genes are weakened, disrupted, and / or deleted to optimize production of indole receptors and / or tryptamine derivatives. A particular target gene to be weakened, disrupted, and / or deleted is a gene encoding a phosphatase that converts psilocybin to psilocin.

[0121] If the host cell is a yeast strain, it may be suitable to be modified by weakening, disrupting and / or deleting one or more wild-type genes selected from: a) the pyruvate kinase gene contained in SEQ ID NO:27 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO:27; b) a phosphofructokinase gene comprised in any of SEQ ID NOs: 29 or 31, or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to any of SEQ ID NOs: 29 or 31; c) a transporter gene comprised in SEQ ID NO: 33 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 33; d) a DL-glycerol-3-phosphate phosphatase gene contained in SEQ ID NO: 34 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 34; e) a tryptophan 2,3-dioxygenase gene contained in SEQ ID NO: 35 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 35; f) a cystathionine β-synthase gene contained in SEQ ID NO: 36 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 36; g) a phenylpyruvate decarboxylase gene contained in SEQ ID NO: 37 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 37; h) a pyruvate decarboxylase gene contained in SEQ ID NO: 38 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 38; i) a histone variant H2AZ gene contained in SEQ ID NO:39 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO:39; j) a phosphatase gene comprised in SEQ ID NO: 40 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 40; k) a inhibitory acid phosphatase gene comprised in any of SEQ ID NOs: 41, 42 or 43, or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to any of SEQ ID NOs: 41, 42 or 43; l) a constitutively expressed acid phosphatase gene comprised in SEQ ID NO: 44 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 44; m) a sterol reductase gene contained in SEQ ID NO: 183 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 183; and / or n) S-adenosylmethionine decarboxylase gene contained in SEQ ID NO: 184 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 184.

[0122] If the host cell is a yeast strain, it may also be suitable to be modified to overexpress one or more wild-type genes selected from the NADH kinase gene contained in SEQ ID NO: 185 or any of its paralogs or orthologs having at least 70%, such as at least 75%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 99%, for example 100% identity to SEQ ID NO: 185.

[0123] The invention also provides a cell culture comprising a host cell of the invention and a growth medium. Suitable growth media for host cells such as plant cell lines, filamentous fungi and / or yeasts are known in the art.

[0124] Nucleotide Constructs In a further aspect, the present invention provides a polynucleotide construct comprising a polynucleotide sequence encoding the (?) of the present invention operably linked to one or more control sequences heterologous to the replacement enzyme-encoding polynucleotide.

[0125] Polynucleotides can be manipulated in various ways to allow them to express polypeptides. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide before it is inserted into the expression vector. The techniques of modifying polynucleotides using recombinant DNA methods are well known in the art.

[0126] The control sequence may be a promoter, which is a polynucleotide that is recognized by a host cell for the expression of a polynucleotide. The promoter comprises a transcriptional control sequence that mediates the expression of a polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutants, truncated, and hybrid promoters, and may be obtained from a gene that encodes a homologous or heterologous extracellular or intracellular polypeptide to the host cell. The promoter may be an inducible promoter. For example, promoters useful for expression in fungi, including yeast, are known in the art.

[0127] The control sequence may also be a transcription terminator, which is a polynucleotide that is recognized by a host cell for terminating transcription.The terminator is operably linked to the 3'-end of the polynucleotide that codes for a polypeptide.Any terminator that is functional in a host cell may be used.For example, the terminator that is useful for expression in fungi, including yeast, is known in the art.

[0128] A regulatory sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene that enhances expression of the gene.

[0129] The control sequence may also be a leader, which is a non-translated region of an mRNA that is important for translation by a host cell. The leader is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used. For example, leaders that are useful for expression in fungi, including yeast, are known in the art.

[0130] The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3'-end of a polynucleotide, which, when transcribed, is recognized by host cell as a signal for adding polyadenosine residues to transcribed mRNA.Any polyadenylation sequence that is functional in host cell may be used.For example, polyadenylation sequences useful for expression in fungi, including yeast, are known in the art.

[0131] It may also be desirable to add regulatory sequences that regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.

[0132] In a further aspect, the present invention provides an expression vector comprising the polynucleotide construct of the present invention. In addition to the polynucleotide construct of the present invention, various nucleotide sequences can be ligated together to produce a recombinant expression vector, which contains one or more convenient restriction enzyme sites, allowing the insertion or substitution of a polynucleotide sequence encoding a relevant polypeptide at such site. The recombinant expression vector can be any vector (e.g., a plasmid or virus or chromosome) that can be conveniently subjected to recombinant DNA techniques and can result in the expression of a relevant polypeptide-encoding polynucleotide. The choice of vector will usually depend on the compatibility of the vector with the host cell into which it is introduced. The vector can be a linear or closed circular plasmid. The vector can be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, and can be, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain some means for ensuring autonomous replication. Alternatively, the nucleotide construct can be integrated into the genome when introduced into a host cell and replicated together with the chromosome into which it is integrated. Additionally, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. A vector may contain one or more selectable markers that allow easy selection of cells that have been transformed, transduced, transduced, etc. A selectable marker is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0133] The vector preferably contains elements that allow the vector to integrate into the genome of the host cell or to autonomously replicate in the cell independent of the genome. For integration into the host cell genome, the vector may rely on a polynucleotide encoding a polypeptide or on any other element of the vector to integrate into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration by homologous recombination into the genome of the host cell at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integration element should contain a sufficient number of nucleic acids, such as, for example, 35-10,000 base pairs, for example, 100-10,000 base pairs, for example, 400-10,000 base pairs, and for example, 800-10,000 base pairs, with a high degree of sequence identity to the corresponding target sequence to increase the establishment of homologous recombination. The integration element may be any sequence that is homologous to the target sequence in the genome of the host cell. Furthermore, the integration element may be a non-coding or coding polynucleotide. Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0134] The origin of replication may be any plasmid origin of replication that mediates autonomous replication that functions in a cell. The term "origin of replication" or "plasmid origin of replication" refers to a polynucleotide that allows a plasmid or vector to initiate replication in vivo.

[0135] More than one copy of a polynucleotide encoding a replacement enzyme or other pathway polypeptide of the invention may be inserted into a host cell to increase production of the polypeptide. Increased copy number can be obtained by integrating additional copies of one or more enzyme coding sequences into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, and cells containing an amplified copy of the selectable marker gene (and thereby additional copies of the polynucleotide) can be selected by culturing the cells in the presence of an appropriate selectable agent. The procedures used to ligate the above elements to construct the recombinant expression vectors of the invention are well known to those of skill in the art (see, for example, Green & Sambrook, 2012, Molecular cloning: A laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory, New York, USA).

[0136] Fermentation methods for producing the compounds of the invention. When the method of the invention is carried out wholly or partly by fermenting a cell culture of a genetically modified host cell of the invention, the method claim advantageously further comprises: a) culturing the cell culture described herein under conditions that allow the host cells to produce the tryptamine derivative (I); and b) optionally recovering and / or isolating the tryptamine derivative (I).

[0137] The cell culture can be cultured in a nutrient medium and under conditions suitable for the production of the tryptamine derivatives of the invention and / or for the expansion of cell number using methods known in the art. For example, the culture can be cultured in a suitable medium, under conditions that allow for the growth and / or proliferation of the host cells, and, if necessary, their recovery and / or isolation, by shake flask culture, or by small- or large-scale fermentation (continuous, batch, fed-batch, feed and draw, or solid-state fermentation) in laboratory or industrial fermentors.

[0138] Cultivation can be performed in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media can be obtained from commercial suppliers or prepared according to published recipes (e.g., from the American Type Culture Collection catalog). Selection of an appropriate medium can be based on the choice of host cell and / or based on regulatory constraints for the host cell. Such media are available in the art. Optionally, the medium can contain additional components that favor the transformed expression host over other potentially included microorganisms. Thus, in an embodiment, a suitable nutrient medium includes a carbon source (e.g., glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulolytic biomass hydrolysate, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g., yeast extract, malt extract, peptone, etc.), and an inorganic nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron, etc.).

[0139] The cultivation of the host cells may be carried out for about 0.5 to about 30 days. The cultivation method may be a batch, continuous or fed-batch method, suitably carried out at a temperature in the range of 0 to about 100°C or 0 to 80°C, for example, about 0°C to about 50°C, and / or at a pH of, for example, about 2 to about 10. Preferred fermentation conditions for yeast and filamentous fungi are a temperature in the range of about 25°C to about 55°C, and a pH of about 3 to about 9. Appropriate conditions are usually selected based on the choice of the host cell. Thus, in one embodiment, the method of the present invention further comprises one or more requirements selected from the following: a) culturing a cell culture in a nutrient growth medium; b) culturing cell cultures under aerobic or anaerobic conditions; c) Incubation of cell cultures under agitation; d) Incubation of cell culture medium at a temperature between 25°C and 50°C; e) incubation of cell cultures at a pH between 3 and 9; f) culturing the cell culture for 10 hours to 30 days; and g) Cultivation of cell cultures under fed-batch, repeated fed-batch, continuous, or semi-continuous conditions.

[0140] The fermentation method of the present invention further suitably comprises feeding one or more exogenous indole acceptors or precursors thereof and / or substituent donors to the cell culture.

[0141] The cell culture fluid and / or metabolites contained therein can be recovered and isolated using methods known in the art. For example, the cells and / or metabolites can be recovered from the nutrient medium by conventional methods, including, but not limited to, centrifugation, filtration, spray drying, or freeze drying. In certain embodiments, the method includes a recovery and / or isolation step, which includes separating the liquid phase of the cells or cell culture fluid from the solid phase of the cells or cell culture fluid to obtain a supernatant containing the tryptamine derivative, and subjecting the supernatant to one or more steps selected from the following: a) disrupting host cells and releasing intracellular tryptamine derivatives into the supernatant; b) separating the supernatant from the host cell solid phase, such as by filtration or gravity separation; c) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the tryptamine derivatives produced; d) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the tryptamine derivatives; e) extracting the tryptamine derivative; and f) precipitating the tryptamine derivative by crystallization or evaporation of the solvent in the liquid phase and, optionally, separating the tryptamine derivative by filtration or gravity separation, thereby recovering and / or isolating the tryptamine derivative.

[0142] Fermentation liquid / composition The present invention provides a fermentation broth / composition comprising the cell culture broth of the present invention and the tryptamine derivatives contained therein. In one embodiment, at least 10%, 25%, 50%, such as at least 75%, such as at least 95%, such as at least 99% of the cells of the fermentation broth / composition of the present invention are lysed.Furthermore, in the fermentation broth / composition of the present invention, at least 10%, 25%, 50%, such as at least 75%, such as at least 95%, such as at least 99% of the solid cellular material can be removed and separated from the liquid phase.Furthermore, in addition to the tryptamine derivative, the fermentation broth / composition of the present invention can also contain precursors, products, metabolites of the indole receptor pathway, in particular tryptophan and / or tryptamine, or one or more compounds selected from fermentation trace metals, vitamins, salts, yeast nitrogen base medium, carbon source, YNB, and / or amino acids. In particular, the fermentation broth / composition comprises a concentration of tryptamine derivative of at least 1 mg / kg or mg / L composition, such as at least 5 mg / kg or mg / L, such as at least 10 mg / kg or mg / L, such as at least 20 mg / kg or mg / L, such as at least 50 mg / kg or mg / L, such as at least 100 mg / kg or mg / L, such as at least 500 mg / kg or mg / L, such as at least 1000 mg / kg or mg / L, such as at least 5000 mg / kg or mg / L, such as at least 10000 mg / kg or mg / L, such as at least 50000 mg / kg or mg / L.

[0143] composition In a further aspect, the present invention further provides a composition comprising the fermentation broth / composition of the present invention and one or more carriers, agents, additives and / or excipients. Carriers, agents, additives and / or excipients include formulation additives, stabilizers, bulking agents, etc. The composition may be formulated into a dry solid form, such as a powder, tablet, capsule, hard chewable and / or soft lozenge or gum, using methods known in the art, such as spray drying, spray cooling, freeze drying, flash freezing, granulation, microgranulation, encapsulation or microencapsulation. The composition may also be formulated into a liquid stabilized form, using methods known in the art, such as formulation into a stabilized liquid containing one or more stabilizers, such as sugars and / or polyols (e.g., sugar alcohols) and / or organic acids (e.g., lactic acid).

[0144] Further Aspects In addition to the method and host cell described herein for producing tryptamine derivatives of formula (I), further aspects relate to a method and host cell for producing serotonin (indole receptor), which is useful in the method described herein for producing tryptamine derivatives of formula (I).While the known pathways to serotonin are long, complicated, and require the addition or design of essential cofactors, the present inventors have newly discovered that serotonin can be produced much more efficiently in genetically modified host cells that produce or are supplied with tryptamine, and express both tryptamine 5-hydroxylase, which converts tryptamine to serotonin; and cytochrome P450 reductase, which assists tryptamine 5-hydroxylase in converting tryptamine to serotonin.

[0145] Accordingly, provided herein are genetically modified microbial host cells that produce a serotonin indole receptor that express one or more genes encoding a polypeptide selected from the following: a) one or more genes encoding a polypeptide selected from: i) one or more enzymes that convert glucose to fructose-6-phosphate; ii) fructose-6-phosphate phosphoketolase, which converts fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate; iii) phosphotransacetylase, which converts acetyl phosphate to acetyl-CoA; iv) one or more enzymes that convert fructose-6-phosphate to phosphoenolpyruvate; v) 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase), which converts phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP); vi) one or more enzymes that convert 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimate 3-phosphate; vii) shikimate kinase, which converts shikimate to shikimate-3-phosphate; viii) Chorismate synthase, which converts 5-enolpyruvoylshikimate 3-phosphate to chorismate; ix) anthranilate synthase, which converts chorismate to anthranilate; x) ribose-phosphate pyrophosphokinase, which converts ribose-5-phosphate to phospho-α-D-ribosyl-1-pyrophosphate; xi) anthranilate phosphoribosyltransferase, which converts anthranilate and phospho-α-D-ribosyl-1-pyrophosphate to N-(5-phosphoribosyl)-anthranilic acid; xii) N-(5'-phosphoribosyl)anthranilate isomerase, which converts N-(5-phosphoribosyl)-anthranilate to 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate; xiii) indole-3-glycerol phosphate synthase, which converts 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate to (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate; xiv) tryptophan synthase, which converts (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate and serine to L-tryptophan; and / or xv) tryptophan decarboxylase, which converts L-tryptophan to tryptamine; and b) a heterologous tryptamine 5-hydroxylase that converts tryptamine to serotonin; and c) Heterologous cytochrome P450 reductase, which assists in the conversion of tryptamine to serotonin by tryptamine 5-hydroxylase.

[0146] When the host cell is Saccharomyces cerevisiae, this species contains the wild-type CYP enzyme (Ncp1), which is not functional with heterologous CYP enzymes such as tryptamine 5-hydroxylase.

[0147] It is shown herein that such genetically engineered host cells can efficiently produce serotonin through an alternative biosynthetic pathway not previously reported. This production method offers a significant improvement over the art by being more efficient but also by mitigating the need to produce or exogenously add tetrahydrobiopterin.

[0148] In an important embodiment of a host cell that produces serotonin, a) the fructose-6-phosphate phosphoketolase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the fructose-6-phosphate phosphoketolase contained in SEQ ID NO:2; b) the phosphotransacetylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the phosphotransacetylase contained in SEQ ID NO:4; c) the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase contained in SEQ ID NO:6; d) an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimate 3-phosphate has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the enzyme contained in SEQ ID NO:8; e) the shikimate kinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the shikimate kinase contained in SEQ ID NO: 10; f) the chorismate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the chorismate synthase contained in SEQ ID NO: 12; g) the anthranilate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the anthranilate synthase contained in SEQ ID NO: 14; h) the ribose-phosphate pyrophosphokinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ribose-phosphate pyrophosphokinase contained in SEQ ID NO: 16; i) the anthranilate phosphoribosyltransferase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the anthranilate phosphoribosyltransferase contained in SEQ ID NO: 18; j) the N-(5'-phosphoribosyl)anthranilate isomerase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the N-(5'-phosphoribosyl)anthranilate isomerase contained in SEQ ID NO: 20; k) the indole-3-glycerol synthase phosphate has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the indole-3-glycerol synthase phosphate contained in SEQ ID NO: 22; l) the tryptophan synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan synthase in any of SEQ ID NOs: 24, 60, 62, 64, 66, 68, 180 and / or 182; m) the tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase included in any of SEQ ID NOs: 26, 70, 72, 74, 76, and / or 78; n) the tryptamine 5-hydroxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the tryptamine 5-hydroxylase contained in SEQ ID NO: 96 (OsT5H); and o) The cytochrome P450 reductase has at least 70%, such as at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity to the cytochrome P450 reductase contained in SEQ ID NO: 112 (FoCPR).

[0149] In a host cell producing serotonin, the expressed gene or genes are preferably selected from: a) a gene encoding a fructose-6-phosphate phosphoketolase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the fructose-6-phosphate phosphoketolase-encoding polynucleotide contained in SEQ ID NO:1, or a genomic DNA thereof; b) a gene encoding a phosphotransacetylase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the phosphotransacetylase-encoding polynucleotide contained in SEQ ID NO:3, or its genomic DNA; c) a gene encoding 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase-encoding polynucleotide contained in SEQ ID NO:5, or a genomic DNA thereof; d) a polynucleotide encoding an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimic acid 3-phosphate and a gene encoding an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimic acid 3-phosphate that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical, or a genomic DNA thereof; e) a gene encoding a shikimate kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a shikimate kinase-encoding polynucleotide contained in SEQ ID NO: 9, or a genomic DNA thereof; f) a gene encoding shikimate kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the chorismate synthase encoding polynucleotide contained in SEQ ID NO: 11, or its genomic DNA; g) a gene encoding an anthranilate synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the anthranilate synthase-encoding polynucleotide contained in SEQ ID NO: 13, or a genomic DNA thereof; h) a gene encoding a ribose-phosphate pyrophosphokinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the ribose-phosphate pyrophosphokinase-encoding polynucleotide contained in SEQ ID NO: 15, or a genomic DNA thereof; i) a gene encoding an anthranilate phosphoribosyltransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the anthranilate phosphoribosyltransferase-encoding polynucleotide contained in SEQ ID NO: 17, or a genomic DNA thereof; j) a gene encoding an N-(5'-phosphoribosyl)anthranilic acid isomerase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the N-(5'-phosphoribosyl)anthranilic acid isomerase-encoding polynucleotide contained in SEQ ID NO: 19, or a genomic DNA thereof; k) a gene encoding an indole-3-glycerol synthase phosphate that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the indole-3-glycerol synthase phosphate-encoding polynucleotide contained in SEQ ID NO: 21, or a genomic DNA thereof; l) a gene encoding a tryptophan synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptophan synthase-encoding polynucleotide comprised in any of SEQ ID NOs: 23, 59, 61, 63, 65, 67, 179, and / or 181, or a genomic DNA thereof; m) tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase encoding genes or genomic DNA thereof that are at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase encoding polynucleotide included in any of SEQ ID NOs: 25, 69, 71, 73, 75, and / or 77; n) a gene encoding tryptamine 5-hydroxylase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the tryptamine 5-hydroxylase-encoding polynucleotide contained in SEQ ID NO: 96, or a genomic DNA thereof; o) a gene encoding a cytochrome P450 reductase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the cytochrome P450 reductase-encoding polynucleotide contained in SEQ ID NO: 111, or a genomic DNA thereof;

[0150] When the host cell producing serotonin is a yeast strain such as Saccharomyces cerevisiae, the host cell is preferably further modified to attenuate, disrupt and / or delete one or more wild-type genes selected from the following: a) the pyruvate kinase gene contained in SEQ ID NO:27 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO:27; b) a phosphofructokinase gene comprised in any of SEQ ID NOs: 29 and / or 31, or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to any of SEQ ID NOs: 29 and / or 31; c) a transporter gene comprised in SEQ ID NO: 33 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 33; d) the DL-glycerol-3-phosphate phosphatase gene contained in SEQ ID NO: 34 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 34; and / or e) a tryptophan 2,3-dioxygenase gene contained in SEQ ID NO: 35 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 35; f) a cystathionine β-synthase gene contained in SEQ ID NO: 36 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 36; g) a phenylpyruvate decarboxylase gene contained in SEQ ID NO: 37 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 37; h) the pyruvate decarboxylase gene contained in SEQ ID NO: 38 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 38; and / or i) A histone variant H2AZ gene contained in SEQ ID NO: 39 or any of its paralogs or orthologs having at least 70%, such as at least 75%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 99%, for example 100% identity to SEQ ID NO: 39.

[0151] In such a yeast host cell, it is also preferably further modified to overexpress one or more wild type genes selected from the NADH kinase gene contained in SEQ ID NO: 185 or any of its paralogs or orthologs having at least 70%, such as at least 75%, for example at least 80%, such as at least 90%, for example at least 95%, such as at least 99%, for example 100% identity to SEQ ID NO: 185.

[0152] In yet a further aspect, provided herein is a cell culture comprising a host cell producing serotonin (indole receptor) and a growth medium.

[0153] In yet a further aspect, provided herein is a method of producing serotonin (indole receptor) comprising: a) culturing the cell culture under conditions that permit host cell production of serotonin; and b) optionally recovering and / or isolating the serotonin.

[0154] In further embodiments, such methods include one or more features selected from the following: a) culturing a cell culture in a nutrient growth medium; b) culturing cell cultures under aerobic or anaerobic conditions; c) Incubation of cell cultures under agitation; d) Incubation of cell culture medium at a temperature between 25°C and 50°C; e) incubation of cell cultures at a pH between 3 and 9; f) culturing the cell culture for 10 hours to 30 days; and g) Cultivation of cell cultures under fed-batch, repeated fed-batch, continuous, or semi-continuous conditions.

[0155] The method may also include providing one or more exogenous precursors of the serotonin pathway to the cell culture.

[0156] The recovery and / or isolation step may comprise separating the liquid phase of the host cells or cell culture from the solid phase of the cells or cell culture and obtaining a supernatant containing serotonin by one or more steps selected from the following: a) disrupting host cells and releasing intracellular serotonin indole receptors into the supernatant; b) separating the supernatant from the host cell solid phase, such as by filtration or gravity separation; c) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the serotonin produced; d) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the serotonin; e) extracting serotonin; and f) Precipitating the serotonin indole receptor by crystallization or evaporation of the liquid phase solvent and, optionally, isolating the serotonin by filtration or gravity separation, thereby recovering and / or isolating the serotonin.

[0157] In yet another aspect, the present invention provides a fermentation liquid / composition that comprises serotonin in a cell culture or growth medium.In such fermentation liquid, at least 50%, for example, at least 75%, for example, at least 95%, for example, at least 99% of the host cells can be destroyed, and further, at least 50%, for example, at least 75%, for example, at least 95%, for example, at least 99% of the solid cell material can be separated from the liquid.Furthermore, in addition to serotonin, the fermentation liquid / composition of the present invention can also comprise precursors, products, metabolites of the serotonin pathway, in particular tryptophan and / or tryptamine, or one or more compounds selected from fermentation trace metals, vitamins, salts, yeast nitrogen base medium, carbon source, YNB, and / or amino acids. In particular, the fermentation broth / composition comprises a concentration of serotonin of at least 1 mg / kg or mg / L composition, such as at least 5 mg / kg or mg / L, such as at least 10 mg / kg or mg / L, such as at least 20 mg / kg or mg / L, such as at least 50 mg / kg or mg / L, such as at least 100 mg / kg or mg / L, such as at least 500 mg / kg or mg / L, such as at least 1000 mg / kg or mg / L, such as at least 5000 mg / kg or mg / L, such as at least 10000 mg / kg or mg / L, such as at least 50000 mg / kg or mg / L.

[0158] In a further aspect, provided herein is a fermentation broth / composition of the present invention and one or more carriers, agents, additives and / or excipients. Carriers, agents, additives and / or excipients include formulation additives, stabilizers, bulking agents, etc. The composition can be formulated into a dry solid form, such as a powder, tablet, capsule, hard chewable and / or soft lozenge or gum, using methods known in the art, such as spray drying, spray cooling, freeze drying, flash freezing, granulation, microgranulation, encapsulation or microencapsulation. The composition can also be formulated into a liquid stabilized form, using methods known in the art, such as formulation into a stabilized liquid containing one or more stabilizers, such as sugars and / or polyols (e.g., sugar alcohols) and / or organic acids (e.g., lactic acid).

[0159] In addition to the methods and host cells described herein for producing tryptamine derivatives of formula (I), further aspects relate to methods and host cells for producing psilocybin (indole receptor), which are useful in the methods described herein, e.g., for the manufacture of tryptamine derivatives of formula (I).

[0160] Accordingly, provided herein are psilocybin-producing genetically modified microbial host cells that express one or more genes encoding a polypeptide selected from the following: a) one or more genes encoding a polypeptide selected from: i) one or more enzymes that convert glucose to fructose-6-phosphate; ii) fructose-6-phosphate phosphoketolase, which converts fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate; iii) phosphotransacetylase, which converts acetyl phosphate to acetyl-CoA; iv) one or more enzymes that convert fructose-6-phosphate to phosphoenolpyruvate; v) 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase), which converts phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP); vi) one or more enzymes that convert 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimate 3-phosphate; vii) shikimate kinase, which converts shikimate to shikimate-3-phosphate; viii) Chorismate synthase, which converts 5-enolpyruvoylshikimate 3-phosphate to chorismate; ix) anthranilate synthase, which converts chorismate to anthranilate; x) ribose-phosphate pyrophosphokinase, which converts ribose-5-phosphate to phospho-α-D-ribosyl-1-pyrophosphate; xi) anthranilate phosphoribosyltransferase, which converts anthranilate and phospho-α-D-ribosyl-1-pyrophosphate to N-(5-phosphoribosyl)-anthranilic acid; xii) N-(5'-phosphoribosyl)anthranilate isomerase, which converts N-(5-phosphoribosyl)-anthranilate to 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate; xiii) indole-3-glycerol phosphate synthase, which converts 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate to (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate; xiv) tryptophan synthase, which converts (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate and serine to L-tryptophan; and / or xv) tryptophan decarboxylase, which converts L-tryptophan to tryptamine; and b) tryptamine 4-hydroxylase, which converts tryptamine to 4-hydroxytryptamine; c) cytochrome P450, which assists tryptamine 4-hydroxylase in converting tryptamine to 4-hydroxytryptamine; d) cytochrome b5, which assists tryptamine 4-hydroxylase in converting tryptamine to 4-hydroxytryptamine; e) 4-hydroxytryptamine kinase, which converts 4-hydroxytryptamine to norbaeocystin; and f) psilocybin synthase, which converts norbeocystin to psilocybin. It is shown herein that such genetically engineered host cells are capable of efficiently producing psilocybin through an alternative biosynthetic pathway not previously reported, and that this production method represents a significant improvement over the art due to its greater efficiency.

[0161] In important embodiments of the psilocybin producing host cell, the host cell comprises: a) the fructose-6-phosphate phosphoketolase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the fructose-6-phosphate phosphoketolase contained in SEQ ID NO:2; b) the phosphotransacetylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the phosphotransacetylase contained in SEQ ID NO:4; c) the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase contained in SEQ ID NO:6; d) an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimate 3-phosphate has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the enzyme contained in SEQ ID NO:8; e) the shikimate kinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the shikimate kinase contained in SEQ ID NO: 10; f) the chorismate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the chorismate synthase contained in SEQ ID NO: 12; g) the anthranilate synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the anthranilate synthase contained in SEQ ID NO: 14; h) the ribose-phosphate pyrophosphokinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ribose-phosphate pyrophosphokinase contained in SEQ ID NO: 16; i) the anthranilate phosphoribosyltransferase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the anthranilate phosphoribosyltransferase contained in SEQ ID NO: 18; j) the N-(5'-phosphoribosyl)anthranilate isomerase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the N-(5'-phosphoribosyl)anthranilate isomerase contained in SEQ ID NO: 20; k) the indole-3-glycerol synthase phosphate has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the indole-3-glycerol synthase phosphate contained in SEQ ID NO: 22; l) the tryptophan synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan synthase in any of SEQ ID NOs: 24, 60, 62, 64, 66, 68, 180 and / or 182; m) the tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase included in any of SEQ ID NOs: 26, 70, 72, 74, 76, and / or 78; n) the tryptamine 4-hydroxylase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a tryptamine 4-hydroxylase in any of SEQ ID NOs: 94 and / or 88; o) the cytochrome P450 reductase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a cytochrome P450 reductase included in any of SEQ ID NOs: 106 and / or 102; p) the cytochrome b5 has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the cytochrome b5 contained in SEQ ID NO: 254; q) the 4-hydroxytryptamine kinase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a 4-hydroxytryptamine kinase contained in any of SEQ ID NOs: 160 or 156; and r) the psilocybin synthase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to a psilocybin synthase contained in any of SEQ ID NOs: 128 or 124.

[0162] In a host cell producing psilocybin, the one or more expressed genes are preferably selected from: a) a gene encoding a fructose-6-phosphate phosphoketolase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the fructose-6-phosphate phosphoketolase-encoding polynucleotide contained in SEQ ID NO:1, or a genomic DNA thereof; b) a gene encoding a phosphotransacetylase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the phosphotransacetylase-encoding polynucleotide contained in SEQ ID NO:3, or its genomic DNA; c) a gene encoding 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase-encoding polynucleotide contained in SEQ ID NO:5, or a genomic DNA thereof; d) a polynucleotide encoding an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimic acid 3-phosphate and a gene encoding an enzyme that converts 3-deoxy-D-arabinoheptulosonic acid-7-phosphate to 5-enolpyruvoylshikimic acid 3-phosphate that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical, or a genomic DNA thereof; e) a gene encoding a shikimate kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a shikimate kinase-encoding polynucleotide contained in SEQ ID NO: 9, or a genomic DNA thereof; f) a gene encoding shikimate kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the chorismate synthase encoding polynucleotide contained in SEQ ID NO: 11, or its genomic DNA; g) a gene encoding an anthranilate synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the anthranilate synthase-encoding polynucleotide contained in SEQ ID NO: 13, or a genomic DNA thereof; h) a gene encoding a ribose-phosphate pyrophosphokinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the ribose-phosphate pyrophosphokinase-encoding polynucleotide contained in SEQ ID NO: 15, or a genomic DNA thereof; i) a gene encoding an anthranilate phosphoribosyltransferase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the anthranilate phosphoribosyltransferase-encoding polynucleotide contained in SEQ ID NO: 17, or a genomic DNA thereof; j) a gene encoding an N-(5'-phosphoribosyl)anthranilic acid isomerase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the N-(5'-phosphoribosyl)anthranilic acid isomerase-encoding polynucleotide contained in SEQ ID NO: 19, or a genomic DNA thereof; k) a gene encoding an indole-3-glycerol synthase phosphate that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to the indole-3-glycerol synthase phosphate-encoding polynucleotide contained in SEQ ID NO: 21, or a genomic DNA thereof; l) a gene encoding a tryptophan synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptophan synthase-encoding polynucleotide comprised in any of SEQ ID NOs: 23, 59, 61, 63, 65, 67, 179, and / or 181, or a genomic DNA thereof; m) tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase encoding genes or genomic DNA thereof that are at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase encoding polynucleotide included in any of SEQ ID NOs: 25, 69, 71, 73, 75, and / or 77; n) a gene encoding a tryptamine 4-hydroxylase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a tryptamine 4-hydroxylase-encoding polynucleotide included in any of SEQ ID NOs: 93 and / or 87; o) a gene encoding a cytochrome P450 reductase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a cytochrome P450 reductase comprised in any of SEQ ID NOs: 105 and / or 101; p) a gene encoding a cytochrome b5 that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a cytochrome b5-encoding polynucleotide contained in SEQ ID NO: 253; q) a gene encoding a 4-hydroxytryptamine kinase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a 4-hydroxytryptamine kinase-encoding polynucleotide included in SEQ ID NO: 159 and / or 155; and / or r) a gene encoding a psilocybin synthase that is at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identical to a psilocybin synthase-encoding polynucleotide comprised in any of SEQ ID NOs: 127 and / or 123.

[0163] When the psilocybin producing host cell is a yeast strain such as Saccharomyces cerevisiae, the host cell is preferably further modified to attenuate, disrupt and / or delete one or more wild type genes selected from the following: a) the pyruvate kinase gene contained in SEQ ID NO:27 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO:27; b) a phosphofructokinase gene comprised in any of SEQ ID NOs: 29 and / or 31, or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to any of SEQ ID NOs: 29 and / or 31; c) a transporter gene comprised in SEQ ID NO: 33 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 33; d) the DL-glycerol-3-phosphate phosphatase gene contained in SEQ ID NO: 34 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 34; and / or e) a tryptophan 2,3-dioxygenase gene contained in SEQ ID NO: 35 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 35; f) a cystathionine β-synthase gene contained in SEQ ID NO: 36 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 36; g) a phenylpyruvate decarboxylase gene contained in SEQ ID NO: 37 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 37; h) a pyruvate decarboxylase gene contained in SEQ ID NO: 38 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 38; i) a histone variant H2AZ gene contained in SEQ ID NO:39 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO:39; j) a phosphatase gene comprised in SEQ ID NO: 40 or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to SEQ ID NO: 40; k) a inhibitory acid phosphatase gene comprised in any of SEQ ID NOs: 41, 42 or 43, and / or any of its paralogs or orthologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to any of SEQ ID NOs: 41, 42 and / or 43; and / or l) A constitutively expressed acid phosphatase gene contained in SEQ ID NO: 44 and / or any of its paralogs or orthologs having at least 70%, such as at least 75%, for example at least 80%, such as at least 90%, for example at least 95%, for example at least 99%, for example 100% identity to SEQ ID NO: 44.

[0164] Attenuating, disrupting and / or deleting one or more of the wild-type phosphatase, repressible acid phosphatase and / or constitutively expressed acid phosphatase genes is particularly useful because expression of these phosphatases has been shown to convert tryptamine from psilocybin to psilocin.

[0165] In such a yeast host cell, it is also preferably further modified to overexpress one or more wild type genes selected from the NADH kinase gene contained in SEQ ID NO: 185 or any of its paralogs or orthologs having at least 70%, such as at least 75%, for example at least 80%, such as at least 90%, for example at least 95%, such as at least 99%, for example 100% identity to SEQ ID NO: 185.

[0166] In yet a further aspect, provided herein is a cell culture comprising a host cell that produces psilocybin (indole receptor) and a growth medium.

[0167] In yet a further aspect, provided herein is a method of producing psilocybin (indole receptor) comprising: c) culturing the cell culture under conditions that permit host cells to produce psilocybin; and d) recovering and / or isolating the psilocybin, as appropriate.

[0168] In further embodiments, such methods include one or more features selected from the following: h) culturing the cell culture in a nutrient growth medium; i) Cultivation of cell cultures under aerobic or anaerobic conditions; j) culturing cell cultures under agitation; k) Incubation of cell cultures at temperatures between 25°C and 50°C; l) incubation of cell cultures at a pH between 3 and 9; m) culturing the cell culture for 10 hours to 30 days; and n) Cultivation of cell cultures under fed-batch, repeated fed-batch, continuous, or semi-continuous conditions.

[0169] The method may also include providing one or more exogenous precursors of the psilocybin pathway to the cell culture.

[0170] The recovery and / or isolation step may include separating the liquid phase of the host cells or cell culture from a solid phase of the cells or cell culture and obtaining a supernatant containing psilocybin by one or more steps selected from the following: g) disrupting the host cells and releasing the intracellular psilocybin indole receptors into the supernatant; h) separating the supernatant from the host cell solid phase, such as by filtration or gravity separation; i) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the psilocybin produced; j) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the psilocybin; k) extracting psilocybin; and l) precipitating the psilocybin indole receptor by crystallization or evaporation of the liquid phase solvent and, optionally, separating the psilocybin by filtration or gravity separation, thereby recovering and / or isolating the psilocybin.

[0171] In yet another aspect, the present invention provides a fermentation liquid / composition that comprises psilocybin in a cell culture or growth medium.In such fermentation liquid, at least 50%, for example, at least 75%, for example, at least 95%, for example, at least 99% of the host cells can be destroyed, and further, at least 50%, for example, at least 75%, for example, at least 95%, for example, at least 99% of the solid cell material can be separated from the liquid.Furthermore, in addition to psilocybin, the fermentation liquid / composition of the present invention can also comprise precursors, products, metabolites of the psilocybin pathway, in particular tryptophan and / or tryptamine, or one or more compounds selected from fermentation trace metals, vitamins, salts, yeast nitrogen base medium, carbon source, YNB, and / or amino acids. In particular, the fermentation broth / composition comprises a concentration of psilocybin of at least 1 mg / kg or mg / L composition, such as at least 5 mg / kg or mg / L, such as at least 10 mg / kg or mg / L, such as at least 20 mg / kg or mg / L, such as at least 50 mg / kg or mg / L, such as at least 100 mg / kg or mg / L, such as at least 500 mg / kg or mg / L, such as at least 1000 mg / kg or mg / L, such as at least 5000 mg / kg or mg / L, such as at least 10000 mg / kg or mg / L, such as at least 50000 mg / kg or mg / L.

[0172] In a further aspect, provided herein is a fermentation broth / composition of the present invention and one or more carriers, agents, additives and / or excipients. Carriers, agents, additives and / or excipients include formulation additives, stabilizers, bulking agents, etc. The composition can be formulated into a dry solid form, such as a powder, tablet, capsule, hard chewable and / or soft lozenge or gum, using methods known in the art, such as spray drying, spray cooling, freeze drying, flash freezing, granulation, microgranulation, encapsulation or microencapsulation. The composition can also be formulated into a liquid stabilized form, using methods known in the art, such as formulation into a stabilized liquid containing one or more stabilizers, such as sugars and / or polyols (e.g., sugar alcohols) and / or organic acids (e.g., lactic acid).

[0173] array This application contains a sequence listing generated in PatentIn type 3.5.1, which has also been submitted electronically in ST25 format, which is hereby incorporated by reference in its entirety. For further reference, the following sequences are included in this application: [Table 1] TIFF2024520057000011.tif227159TIFF2024520057000012.tif230159TIFF2024520057000013.tif227159TIFF2024520057000014.tif235159TIFF2024520057000015.tif226159TIFF2024520057000016.tif231159TIFF2024520057000017.tif226159TIFF2024520057000018.tif234159TIFF2024520057000019.tif225159TIFF2024520057000020.tif233159TIFF2024520057000021.tif225159TIFF2024520057000022.tif225159TIFF2024520057000023.tif235159TIFF2024520057000024.tif235159TIFF2024520057000025.tif238159TIFF2024520057000026.tif19159

[0174] References Gietz,R.D.,& Woods,R.A.(2002).Transformation of yeast by lithium acetate / single-stranded carrier DNA / polyethylene glycol method.Methods in Enzymology,350(2001),87-96.https: / / doi.org / 10.1016 / S0076-6879(02)50957-5. Krengel,F.,Mijangos,M.V,Reyes-lezama,M.,& Reyes-chilpa,R.(2019).Extraction and Conversion Studies of the Antiaddictive Alkaloids Coronaridine,Ibogamine ,Voacangine ,and Ibogaine from Two Mexican Tabernaemontana Species( Apocynaceae ).https: / / doi.org / 10.1002 / cbdv.201900175. Maury,J.,Germann,S.M.,Baallal Jacobsen,S.A.,Jensen,N.B.,Kildegaard,K.R.,Herrgard,M.J.,Schneider,K.,Koza,A.,Forster,J.,Nielsen,J.,& Borodina,I.(2016).EasyCloneMulti:A set of vectors for simultaneous and multiple genomic integrations in saccharomyces cerevisiae.PLoS ONE,11(3),1-22.https: / / doi.org / 10.1371 / journal.pone.0150394. Mikkelsen,M.D.,Buron,L.D.,Salomonsen,B.,Olsen,C.E.,Hansen,B.G.,Mortensen,U.H.,& Halkier,B.A.(2012).Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform.Metabolic Engineering,14(2),104-111.https: / / doi.org / 10.1016 / j.ymben.2012.01.006. Janis Fricke, DA(2019).Enzymatic Route toward 6-Methylated Baeocystin and Psilocybin.ChemBioChem,2824-2829. Michael E.Lee,WC(2015).A Highly Characterized Yeast Toolkit for Modular,Multipart Assembly.ACS Synthetic Biology,975-986.

[0175] Working Example material and method material Chemicals used in the examples herein, for example buffers and substrates, are commercially available products of at least reagent grade.

[0176] Background strain BY4741 is a common strain of Saccharomyces cerevisiae derived from S288C and is available from the American Type Culture Collection (ATCC#200885). DH5α and XJb(DE3) are common strains of E. coli available from, for example, Zymo Research.

[0177] Example 1 - Construction of genetically modified Saccharomyces cerevisiae strains for the de novo production of tryptamine and substituted tryptamine derivatives Part 1 De novo production of high levels of tryptamine and substituted tryptamine derivatives in Saccharomyces cerevisiae was achieved by introducing a heterologous tryptophan decarboxylase (CrTdc) that allows the conversion of the wild-type Saccharomyces cerevisiae metabolite tryptophan to tryptamine. To further increase the flux in the direction of tryptamine, a series of wild-type genes were deleted to reduce the diversion of flux from the target pathway and, at the same time, remove the control of the tryptophan biosynthetic pathway. Wild-type Saccharomyces cerevisiae genes involved in the biosynthetic pathway were overexpressed as well as feedback insensitive mutants. Furthermore, a heterologous fructose-6-phosphate "bypass" was introduced to divert flux from central glycolysis in the direction of erythrose 4-phosphate. Finally, to divert carbon flow from central glycolysis to the shikimate pathway, downregulation of CDC19, PFK1 and PFK2 was performed by replacing the wild-type promoter with a truncated version of the URA3 promoter. Truncation of the URA3 promoter resulted in reduced promoter activity, thereby achieving reduced enzymatic activity of the target genes. A general scheme of the tryptamine biosynthetic pathway is shown in Figure 1.

[0178] Genes for the tryptamine biosynthetic pathway were integrated into defined genomic "landing pads" using custom-made overexpression plasmids similar to the system reported by (Mikkelsen et al., 2012). Linear integration fragments were generated by NotI digestion of custom-designed plasmids containing strong constitutive S. cerevisiae promoters and terminators, and flanked by upstream and downstream homology regions to facilitate assembly by homologous recombination. To facilitate assembly of multiple integration plasmids at a single genomic locus, upstream and downstream homology arms were designed and, after NotI digestion (New England Bio Labs Inc.), the linear integration fragments can be recombined into a single linear integration fragment and integrated into the target genomic location. To select transformants that have successfully integrated the desired fragment, endonucleases such as MAD7 can be used as described above, or a selection marker such as LEU2 can be incorporated into the linear integration fragment and transformed into a S. cerevisiae strain that is auxotrophic for leucine as known in the art. To reduce the occurrence of false positives, the selection marker can be split across two linear integration fragments, such as Rec1 and Rec2, such that a functional LEU2 selection marker can be generated only upon successful homologous recombination of the Rec1 and Rec2 integration fragments, as shown in Figure 2.

[0179] The genes were codon-optimized for expression in yeast, synthesized, and inserted into custom integration plasmids by Twist Biosciences (Table 1). After linearization by restriction digestion with NotI (New England Bio Labs Inc.), the plasmids were transformed into Saccharomyces cerevisiae according to (Gietz & Woods, 2002). Transformants were plated on selective media. [Table 2] TIFF2024520057000028.tif66159

[0180] Part 2 As an alternative, we constructed integrative plasmids, also based on the MoClo system. Here, a DNA part library containing the promoter, terminator, gene of interest, and overlapping homology arms (for genomic integration and homologous recombination with the adjacent integrative cassette) was inserted into an entry vector using a standard type II BsmbI restriction cloning reaction. To construct a full integrative expression cassette overexpressing the gene of interest with a strong constitutive promoter and terminator as well as overlapping integrative homology arms, the entry part plasmids were mixed in a one-pot type II BsaI restriction cloning reaction to generate the final integrative plasmid. As described above, a predefined genomic "landing pad" was used as the integration site. To select for transformants with a correctly integrated cassette, a counter selection marker (e.g., URA3 and AmdS) was integrated along with the expression cassette of interest. Since the marker cassette was split into two halves and encoded in the overlapping homology arms between the integrative cassettes, two or more sets of precise integrations should be required for functional expression of the marker. To facilitate counterselection of the marker (by plating on 5-FOA or fluoroacetamide), the marker cassette was flanked by 500 bp of homologous sequences. A list of MoClo-based yeast integrative plasmids used for overproduction of tryptamine and other substituted tryptamine derivatives is shown in Table 2. [Table 3]

[0181] Wild-type Saccharomyces cerevisiae genes were deleted by marker replacement in Saccharomyces cerevisiae strains, which, for example, BY4741, are auxotrophic for essential amino acids. Deletion cassettes were prepared by PCR amplification of auxotrophic marker cassettes with primers containing upstream and downstream homology to the coding region of the gene of interest to be deleted. Subsequent transformation of the integration cassette into a Saccharomyces cerevisiae strain and selection on medium lacking the required amino acid resulted in integration of the marker cassette at the gene of interest. In some cases, additional direct repeat sequences were introduced flanking the marker cassette to counterselect the marker for repeated use. For example, a Ura3 cassette was flanked by direct repeat sequences so that the marker could be looped out upon incubation of the Saccharomyces cerevisiae strain on 5-fluoroorotic acid (5-FOA)-containing medium. A diagram showing PCR amplification of knockout cassettes is shown in Figure 3. Finally, in some cases, an endonuclease such as MAD7 was used to induce a DNA break at the location of the gene of interest, removing the gene by replacement with a DNA cassette consisting of the DNA flanking the gene of interest. A list of the gene deletion cassettes used in the examples that follow is shown in Table 3. [Table 4] To produce novel substituted tryptamine derivatives in Saccharomyces cerevisiae, the derivatizing enzyme was introduced into a strain producing an indole acceptor such as tryptamine or other substituted tryptamine. To achieve this, the gene encoding the derivatizing enzyme was codon-optimized, synthesized, and cloned by Twist into a centromeric plasmid derived from p415TEF containing a LEU2 auxotrophic marker. The expression plasmid was then transformed into a Saccharomyces cerevisiae strain that is auxotrophic for leucine. Alternatively, the gene was integrated into a predefined genomic landing pad using the integration cassette described above. The derivatizing enzyme expression plasmids are shown in Tables 1, 2, and 4. Plasmid DNA or DNA for integration into the genome was introduced into Saccharomyces cerevisiae using the LiAc method known in the art.

[0182] Example 2 - Construction of genetically modified Saccharomyces cerevisiae strains for the production of substituted tryptamine derivatives by feeding a substituted indole acceptor Part 1 The Saccharomyces cerevisiae strain producing substituted tryptamine derivatives from substituted indole acceptors was constructed in three steps. In step 1, genes encoding the enzymes catalyzing the conversion of (substituted) indole to (substituted) tryptophan (tryptophan synthase and tryptophan synthase β subunit) were codon-optimized, synthesized, and cloned by Twist into a p413TEF-derived centromeric plasmid containing a HIS3 auxotrophic marker. The expression plasmid was then transformed into a Saccharomyces cerevisiae strain auxotrophic for histidine. In step 2, genes encoding the enzymes catalyzing the conversion of (substituted) tryptophan to (substituted) tryptamine (tryptophan decarboxylase) were codon-optimized, synthesized, and cloned by Twist into a Ty integration vector with a truncated Ura3 marker as described by (Maury et al., 2016). The galactose-inducible GAL10 promoter was used to drive the expression of these tryptophan decarboxylase genes, so that the enzymes should be active only in galactose-containing medium. The integrated plasmid was then transformed into a Saccharomyces cerevisiae strain auxotrophic for uracil. In step 3, a gene encoding an enzyme (derivatase) that catalyzes the conversion of (substituted) tryptamine to (substituted) tryptamine derivatives was codon-optimized, synthesized, and cloned by Twist into a p415TEF-derived centromeric plasmid containing a LEU2 auxotrophic marker. The expression plasmid was then transformed into a Saccharomyces cerevisiae strain auxotrophic for leucine. Different gene combinations for each of the three steps can be used to produce a series of substituted tryptamine derivatives. [Table 5]

[0183] A list of all Saccharomyces cerevisiae strains constructed according to Examples 1 and 2, together with the corresponding genotypes, is shown in Tables 5 and 6. For simplicity, the constitutive terminators used to overexpress each gene have been omitted, but consist of one of the following strong terminators: tADH1, tCYC1, tSSA1, tENO2, tTDH1, tPGK1, tTDH2, tPGI1. [Table 6]

[0184] Part 2 An additional list of the Saccharomyces cerevisiae strains constructed in Examples 1 and 2 is provided in Table 6. [Table 7] TIFF2024520057000034.tif242159TIFF2024520057000035.tif242159TIFF2024520057000036.tif242159TIFF2024520057000037.tif97159

[0185] Example 3 - Construction of E. coli strains for the production of substituted tryptamine derivatives by feeding a substituted indole acceptor Part 1 An E. coli strain expressing genes that convert exogenously supplied substituted indole acceptors to substituted tryptamine derivatives was constructed as follows. Genes that convert substituted indole to substituted tryptophan and substituted tryptophan to substituted tryptamine were synthesized by Twist and cloned into the pRSFDuet-1 expression plasmid. Transformants were selected by plating on kanamycin-containing medium. Genes that further convert substituted tryptamine to substituted tryptamine derivatives were synthesized by Twist and cloned into a custom-made plasmid vector (pRSGLY, synthesized by GeneArt) using standard restriction ligation with SpeI / XhoI restriction enzyme sites. This custom-made vector contained a LacI operon, an AmpR cassette, an origin of replication and multiple cloning sites flanked by a T7 promoter and terminator. In addition, the 5' end also contained a ribozyme binding site (RBS) and a 6xHis tag for subsequent protein purification. The fully assembled plasmids were transformed into E. coli DH5α or E. coli XJb(DE3) autolytic strains (Zymo Research). The plasmids that convert substituted indoles to substituted tryptamines and substituted tryptamines to substituted tryptamine derivatives are shown in Tables 7 and 8. When the E. coli strains were used to produce and purify enzymes for in vitro reactions, the genes that convert substituted tryptamines to substituted tryptamine derivatives were expressed on themselves to facilitate substituted tryptamine feeding experiments as well as to facilitate in vitro biocatalysis experiments. [Table 8] TIFF2024520057000039.tif42159

[0186] Part 2 Alternatively, genes were synthesized by Twist and cloned as N-terminal His-tagged genes into the pET-28a(+) expression vector. Plasmids were cloned as described above. An additional list of plasmids constructed in Example 3 is shown in Table 8. [Table 9]

[0187] Example 4 - Cultivation of genetically modified Saccharomyces cerevisiae strains for the de novo production of tryptamine and substituted tryptamine derivatives Part 1 The genetically modified yeast strains were pre-cultured in 500 μL of liquid Delft minimal medium containing 20 g / L glucose and related amino acid supplement in 2 mL microtiter plates with air-permeable seals at 30° C. and 280 rpm for 48 h. Then, 10 μL of the yeast pre-culture was transferred to 490 μL of Delft minimal medium containing 20 g / L glucose and related amino acid supplement and cultivated at 30° C. under 280 rpm for 72 h. After cultivation, extracellular metabolites were extracted by mixing 100% methanol with whole cell broth 1:1, vortexing thoroughly, and centrifuging at 4000×g for 5 min. The supernatant was then diluted in Milli-Q water to obtain a final methanol concentration of 12.5% ​​in the samples, which were then analyzed using UHPLC or LC-MS / MS as described in Example 8. When possible, certified analytical standards were used for quantification of tryptamine and tryptamine derivatives.

[0188] Part 2 Alternatively, the genetically modified yeast strains were grown as described above, except that extracellular metabolites were extracted as follows: samples were centrifuged at 4000xg for 5 min, and the supernatant was mixed 1:1 with 100% acetonitrile. Extracted samples were diluted as necessary to ensure that the extracted metabolites were within the calibration range of the analytical method.

[0189] Example 5 - Cultivation of genetically modified Saccharomyces cerevisiae strains for the production of substituted tryptamine derivatives by feeding substituted indoles The genetically engineered yeast strains were pre-cultured for 48 hours at 30°C under 280 rpm in 500 μL of liquid synthetic complete medium containing 20 g / L of glucose-containing combinations of relevant dropouts in 2 mL microtiter plates with air-permeable seals. Then, 10 μL of pre-culture was transferred to 490 μL of synthetic minimal (SD) medium supplemented with relevant amino acids, containing 20 g / L of glucose. In some cases, the cultivation and pre-culture were carried out as described in Example 4. An ethanolic solution of substituted indole was added to the culture medium to obtain a final concentration of 1 mM substituted indole and 2% ethanol, and the strains were cultivated for 72 hours at 30°C under 280 rpm. During this initial cultivation, the genetically modified strains converted biosynthetically produced serine and endogenously supplied substituted indole to substituted tryptophan derivatives. This substituted tryptophan derivative was left inside the genetically modified cells. The culture broth was then centrifuged (3000xg 5 min) and the supernatant was discarded. The pellet was resuspended in 500 μL of SD or Delft medium supplemented with relevant amino acids and 20 g / L galactose to induce expression from the Gal promoter, and the strain was cultivated for an additional 72 h at 30° C. under 280 rpm. During this secondary cultivation, galactose-induced expression of the promiscuous tryptophan decarboxylase converts the substituted tryptophan to its corresponding substituted tryptamine, which can then be freely excreted from the cell. In some cultures, the Saccharomyces cerevisiae strain further contains a derivatization enzyme, which can further convert the produced substituted tryptamine to a substituted tryptamine derivative. After cultivation, the whole cell broth was transferred to a 2 mL screw-cap tube containing glass beads (1 mm diameter) and bead-broken for 45 s at 6.5 m / s with a FastPrep® FP120 cell disrupter (Thermo Savant). Lysed yeast cells were spun down by centrifugation (4000xg for 5 min) and the supernatant was analyzed by UHPLC or LC-MS / MS as described in Example 8. In some cases, whole cell broths were centrifuged at 4000xg for 5 min and the supernatant was extracted and analyzed by UHPLC or LC-MS / MS. Whenever possible, authenticated analytical standards were used to confirm the identity of the produced molecules.

[0190] Example 6 - Cultivation of genetically modified E. coli strains for the production of substituted tryptamine derivatives by feeding substituted indole acceptors E. coli strains were precultured in 500 μL of liquid LB medium supplemented with kanamycin and / or ampicillin, as appropriate, in 2 mL microtiter plates with air-permeable seals at 300 rpm and 37° C. for 24 h. Then, 50 μL of the preculture was transferred to 450 μL of LB medium containing 20 g / L glucose, a polypeptide expression inducer (3 mM arabinose + 0.1 mM IPTG) and an ethanol solution of various substituted indole molecules (1 mM indole, 2% ethanol) and cultured at 300 rpm and 37° C. for 24 h. After cultivation, the whole cell broth was transferred to a 2 mL screw-cap tube containing glass beads (1 mm diameter) and bead-broken for 45 s at 6.5 m / s with a FastPrep® FP120 cell disrupter (Thermo Savant). Lysed E. coli cells were spun down by centrifugation (4000xg for 5 min) and the supernatant was analyzed by UHPLC or LC-MS / MS as described in Example 8. In some cases, whole cell broths were centrifuged at 4000xg for 5 min and the supernatant was extracted and analyzed by UHPLC or LC-MS / MS. Whenever possible, authenticated analytical standards were used to confirm the identity of the produced molecules.

[0191] Example 7 - Assay conditions for biocatalytic production of substituted tryptamine derivatives by feeding indole or tryptamine substrates in an in vitro enzyme assay Part 1 In some cases, production of substituted tryptamine derivatives can be carried out using purified enzymes with the addition of required cofactors and substrates. Preparation and execution of the biocatalytic reaction can be carried out as follows: 5 mL of 2x concentrated LB medium + ampicillin (50 μg / mL) was inoculated with E. coli XJb(DE3) strain expressing the gene of interest and incubated overnight at 30 °C with shaking. The next day, the cell culture was transferred to 500 mL of 2x concentrated LB medium + ampicillin (50 μg / mL) and incubated overnight at 30 °C with shaking. The next day, the cell culture was transferred to 1 L of 2x concentrated LB medium + ampicillin (50 μg / mL) + 3 mM arabinose + 0.1 mM arabinose + 0.1 mM IPTG. The cells were incubated at 20 °C for 24 h with shaking. The next day, the cells were harvested by centrifugation at 6500 x g for 10 min at 4 °C. The cells were resuspended in 20 mL of ice-cold GT buffer (50 mM Tris-HCl pH 7.4 + 1 mM phenylmethanesulfonyl fluoride + 1 cOmplete™ mini, EDTA-free protease inhibitor cocktail tablet (Roche)). The resuspended material was transferred to a 50 mL Falcon tube and kept at -80°C for at least 15 minutes. The Falcon tube was then thawed at room temperature and the following reagents were added as the tube thawed; 2.6 mM MgCl2, 1 mM CaCl2, 1.4 mg / ml DNase solution (Sigma) in 250 μL Milli-Q water. The tube was mixed by gentle inversion and then incubated at 37°C for 5 minutes. 4x binding buffer (10 mL) was then added to the tube (50 mM Tris-HCl pH 7.4, 10 mM imidazole, 500 mM NaCl to a final concentration, and the pH was adjusted to 7.4 with HCl). The mixture was centrifuged at 10000xg for 30 min at 4°C, the supernatant was transferred to a new 50 mL Falcon tube, and centrifuged again at 10000xg for 30 min at 4°C to remove all remaining necrotic cell debris. While the enzyme prep was being centrifuged, 3 mL of HIS-Select (available from Sigma P6611) column material was added to a new tube and washed by adding Milli-Q water to 50 mL, centrifuging at 2000xg for 2 min, and discarding the supernatant. This washing step was repeated. Finally, Milli-Q water was added to the HIS-Select material to approximately 50% of the volume.The collected supernatant from the centrifuged enzyme preparation was transferred through Miracloth (available from Merck Millipore) to the HIS-Select material-containing tube and then incubated at 4° C. for 2 hours with gentle shaking by inversion. After 2 hours, the mixture was centrifuged at 2000×g for 4 minutes at 4° C. and the supernatant was discarded. The remaining HIS-Select material was washed twice with 1× Binding Buffer (50 mM Tris-HCl, 0.5 M NaCl, 10 mM Imidazole, pH 7.4) combined with centrifugation at 2000×g for 4 minutes at 4° C. The HIS-Select material was resuspended in 5 mL of 1× Binding Buffer and transferred to a Poly-Prep® Chromatography Column (available from BioRad, 7311550). The HIS-Select material was kept at 4° C. and washed twice with 1× Binding Buffer by loading the column and dripping it through. Finally, the purified enzyme was eluted from the HIS-Select material by adding 7.5 mL of elution buffer (50 mM Tris-HCl, 500 mM imidazole, pH 7.4) and collecting the flow-through. The enzyme was either used immediately for in vitro enzyme assays or stored in 50% glycerol at -20°C until required.

[0192] In vitro conversion of various (substituted) indoles to (substituted) tryptophans and / or (substituted) tryptophans to (substituted) tryptamines and / or (substituted) tryptamines to (substituted) tryptamine derivatives was carried out according to Table 9. Depending on the substrate, the substrate was dissolved in water, methanol or DMSO. Required cofactors were provided by commercial suppliers (e.g., Sigma). [Table 10]

[0193] The reaction mixture was scaled up or down as required. The reaction mixture was incubated at 30° C. for 24 hours without shaking. Samples were extracted by the addition of ice-cold 100% MeOH to a final concentration of 75% followed by centrifugation at 4000 rpm for 10 minutes. The supernatant was diluted to 12.5% ​​with water prior to analysis as described in Example 8. To confirm the identity of the substituted indoles produced, the expected mass and fragmentation pattern of each detected molecule was confirmed using LC-MS / QTOF as described in Example 8. Quantification of substituted indole production was performed by comparison of peak areas of the indole substrate and substituted indole with certified analytical standards (when available) and, when the substrate was not available, quantification was achieved by comparison with certified analytical standards of the indole substrate. The % conversion of the substrate to substituted indole by the enzyme biocatalysis was calculated by measuring the decrease in substrate and increase in product after 24 hours of incubation.

[0194] Part 2 Alternatively, 5 mL of 2x concentrated LB medium + appropriate antibiotics (50 μg / mL) was inoculated with E. coli XJb(DE3) strain expressing the plasmid of interest and incubated overnight at 30° C. with shaking. The next day, 50 mL of TB medium + antibiotics in a 250 mL baffled flask was inoculated with 0.5 mL of the overnight cell culture and incubated at 30° C. for 3.5 hours. The cell culture was then induced with 3 mM arabinose, 0.1 mM IPTG and incubated at 25° C. for 24 hours with shaking. The next day, cells were harvested by centrifugation at 6500×g for 10 minutes at 4° C. Cells were resuspended in 5 mL of ice-cold protein extraction buffer (50 mM Tris-HCl pH 7.4 + 1 mM phenylmethanesulfonyl fluoride + 1 cOmplete™, mini, EDTA-free protease inhibitor cocktail tablet (Roche)). The resuspended material was transferred to five 1.5 mL Eppendorf tubes and kept at -80°C for at least 15 minutes. The tubes were then thawed at room temperature and as the tubes thawed the following reagents were added; 2.6 mM MgCl2, 1 mM CaCl2, 300 U / mL DNase solution (DENARASE®, C-lecta) and 0.2 mg / mL lysozyme (Sigma) dissolved in Milli-Q water. The tubes were mixed by gentle inversion and then incubated at 37°C for 10-15 minutes. Three volumes of 4x binding buffer were then added to the tubes (50 mM Tris-HCl pH 7.4, 10 mM imidazole, 500 mM NaCl to a final concentration and the pH adjusted to 7.4 with HCl). The mixture was centrifuged at 10,000xg for 30 min at 4°C, and the supernatant was transferred to a new Eppendorf tube and centrifuged again at 10,000xg for 30 min at 4°C to remove any remaining necrotic cell debris. While the enzyme prep was being centrifuged, a HisPur 0.2ml spin column (Thermo Scientific) was prepared as per the manufacturer's instructions. The HisPur column was washed with Milli-Q water and equilibrated twice with 2x resin bed volume of 1xhis binding buffer. The HisPur column was centrifuged at 700xg for 2 min, and the buffer was removed.600 μL of collected supernatant from the centrifuged enzyme preparation was transferred to the HisPur column and then incubated at 4° C. for 30 min with gentle shaking by inversion. After 30 min, unbound proteins were removed by centrifugation at 700×g for 2 min. The remaining 600 μL of collected supernatant from the centrifuged enzyme preparation was loaded onto the HisPur column and incubated again at 4° C. with shaking. Nonspecifically bound other proteins were removed by washing twice with 1× binding buffer (50 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 7.4). The enzyme was resuspended in 200 μl of elution buffer for 2 min and then eluted by centrifugation (700×g, 4° C. for 2 min). The enzyme was used immediately for in vitro enzyme assays or stored in 50% glycerol at −20° C. until required. [Table 11]

[0195] Example 8 - Detection and Quantification Methods for Substituted Tryptamine Derivatives Part 1 A Dionex UltiMate 3000 Quaternary Rapid Separation UHPLC coupled to a Compact micrOTOF-Q mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive ion mode. +LC-MS / QTOF was performed on a dedicated system (Thermo Fisher Scientific, Germering, Germany). Separation was achieved on a Kinetex XB-C18 column (150 × 2.1 mm, 1.7 μm, 100 Å, Phenomenex). For elution, 0.05% (vol / vol) formic acid in HO and 0.05% (vol / vol) formic acid in acetonitrile were employed as mobile phases A and B, respectively. The gradient conditions were as follows: 0.0–1.0 min 2% B; 1.0–24.0 min 2–75% B, 24.0–25.0 min 75–100% B, 25.0–27.5 min 100% B, 27.5–28.0 min 100–2% B, and 28.0–30.0 min 2% B. The mobile phase flow rate was 300 μL / min and the injection volume was 10 μL. The column oven temperature was maintained at 30 °C. UV spectra for each sample were acquired at 220, 230, 240, and 280 nm. The ion spray voltage was maintained at +4500 V. The anhydrous temperature was set at 250 °C and the drying gas flow was set at 8 L / min. Nitrogen was used as the drying, nebulizing, and collision gas. The nebulizing gas was set at 2.5 bar and the collision energy was set at 10 eV. MS spectra were acquired in the range of 50-1000 amu and MS / MS spectra were acquired in the range of 100-800 amu. The sampling rate was 2 Hz. Sodium formate clusters were used for mass calibration. All files were automatically calibrated by post-processing. Data were processed using Bruker Compass DataAnalysis 4.3.

[0196] UHPLC analysis of tryptamine, serotonin, and other substituted tryptamine derivatives was performed on an Agilent 1290 Infinity II LC system (Agilent Technologies, Boblingen, Germany). Separation was achieved on a Kinetex XB-C18 column (100 × 2.1 mm, 1.7 μm, 100 Å, Phenomenex). Isocratic elution with 0.05% (vol / vol) formic acid in H2O was employed with a flow rate of 0.60 ml / min and a run time of 5 min. The column oven temperature was maintained at 35 °C and the injection volume was 5 μL. UV spectra for each sample were acquired at 220 nm. Data were processed using Agilent Openlab CDS Chemstation Rev. C.01.10.

[0197] Part 2 Alternatively, UHPLC analysis of tryptamine, serotonin, and other substituted tryptamine derivatives was performed on an Agilent 1290 Infinity II LC system (Agilent Technologies, Boblingen, Germany). Separation was performed on a Waters ACQUITY UPLC CSH Fluoro-Phenyl Column (100x2.1mm, 1.7μm, 130Å) using gradient elution. For elution, 10mM ammonium acetate in H2O, pH=7 and 10% water in 90% (vol / vol) methanol containing 10mM ammonium acetate were used as mobile phases A and B, respectively. The gradient conditions were as follows: 0.0-5.0 min 5-95% B; 5.0-7.0 min 95% B; 7.0-7.1 min 95-5% B. The flow rate of the mobile phase was 0.400ml / min and the injection volume was 2μL. The column oven temperature was maintained at 35° C. UV spectra for each sample were acquired at 220 and 280 nm. Data were processed using Agilent Openlab CDS Chemstation Rev.C.01.10.

[0198] UHPLC analysis of baeocystin, norbaeocystin, psilocybin and other substituted tryptamine derivatives was performed on an Agilent 1290 Infinity II LC system (Agilent Technologies, Boblingen, Germany). Separation was performed on a Waters ZORBAX RRHD HILIC Plus column (100x2.1mm, 1.8μm, 95Å) using gradient elution. Mobile phase A consisted of 5% H2O in 95% (v / v) acetonitrile containing 10mM ammonium acetate and 10mM formic acid, while mobile phase B consisted of 50% H2O in 50% (v / v) acetonitrile containing 10mM ammonium acetate and 10mM formic acid. The gradient conditions were as follows: 0.0–6.0 min 5% B, 6.0–11.5 min 5–95% B, 11.5–12.0 min 95% B, 12.0–12.1 min 95–5% B, 12.1–12.2 min 5% B. The flow rate of the mobile phase was 0.400 ml / min, and the injection volume was 1 μL. The column oven temperature was maintained at 40 °C. UV spectra for each sample were acquired at 210 and 280 nm. Data were processed using Agilent Openlab CDS Chemstation Rev. C.01.10.

[0199] LC-MS analysis of psilocybin and related derivatives and metabolites was performed as follows. High-resolution LC-MS measurements were performed on a Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific, US) coupled to an Orbitrap Fusion Mass Spectrometer (Thermo Fisher Scientific, US). The UHPLC was equipped with a SeQuant zic-Hilic column (Merck KgaA), 15 cm x 2.1 mm, 3 μm. The temperature was 35 °C and the flow rate was 0.5 mL / min. The system was operated with isocratic elution using a mobile phase consisting of 20% 10 mM ammonium formate (pH 3) and 80% acetonitrile with 0.1% formic acid. Samples were delivered to the MS equipped with a heated electrospray ionization source (HESI) in positive ion mode with the sheath gas set at 50 (au), the auxiliary gas set at 10 (au) and the sweep gas set at 1 (au). Cone and probe temperatures were 325 °C and 350 °C, respectively, and the spray voltage was 3500 V. The scan range was 100-800 Da, with an interscan time of 50 ms. In all cases, certified analytical standards were used for the produced metabolites.

[0200] Example 9 - Chemical stability testing of substituted tryptamine derivatives The chemical stability of substituted tryptamine derivatives was measured under alkaline, acid, oxidative and thermal stress as follows. A 25 mM stock solution of the target tryptamine molecule was prepared in 100% methanol. 15 μL was mixed with 5 μL of 400 mM HCl solution (final pH=1.1), 400 mM NaOH solution (final pH=12.5), 12% H2O2 solution (final concentration 3%) or H2O pH 7.0. Acidic, alkaline and oxidative samples were incubated at 30 °C for 24 h, while the aqueous samples were incubated at 80 °C for 24 h. Controls under ambient conditions were also prepared in the same way, where 15 μL of the molecule was added to 5 μL of H2O pH 7.0 and incubated at 30 °C. After 24 h, the samples were placed on ice and 60 μL of ice-cold 100% methanol was added to each sample. The samples were centrifuged and transferred to HPLC vials for analysis. The remaining concentrate was quantified by comparison with certified analytical standards. The identity of the presence of degradation products was determined by comparison with certified analytical standards.

[0201] Example 10. In vitro production of substituted tryptamine glycosides For in vitro testing of the performance of the glycosyltransferases in glycosylation of substituted tryptamines, purified glycosyltransferases were prepared as described in Example 7 and in vitro enzyme assays were performed as described in Table 11 below. [Table 12]

[0202] The reaction mixture was scaled up or down as necessary. The reaction mixture was incubated at 30° C. for 24 hours without shaking. Extraction and analysis were performed as described for this example (Example 7 and Example 8). To confirm the identity of the substituted tryptamine glycoside produced, the expected mass and fragmentation pattern of each detected molecule was confirmed using LC-MS / QTOF as described above (Example 8). Quantification of substituted tryptamine glycoside production was performed by comparison of peak areas of the tryptamine substrate and substituted tryptamine glycoside with certified analytical standards (when available), and when standards were not available, quantification was achieved by comparison with certified analytical standards of the substituted tryptamine aglycone. The % conversion of the substituted tryptamine substrate to the substituted tryptamine glycoside by the specific glycosyltransferase was calculated by measuring the decrease in substrate and increase in product after 24 hours of incubation. Quantification was also achieved using the UHPLC analytical method described in Example 8.

[0203] Example 11 - De novo high-level production of tryptamine by genetically modified Saccharomyces cerevisiae strains Part 1 A budding yeast strain that produces large amounts of tryptamine was constructed as described above. Tryptamine-producing strains are ideal "mother strains" for introducing derivatizing enzymes for the purpose of de novo production of a series of substituted tryptamine derivatives. Cultivation, extraction, and analysis were carried out as described in Examples 4 and 8 to quantify the amount of tryptamine produced by various genetically modified budding yeast strains shown in Table 12. Overexpression of various heterologous and wild-type budding yeast genes resulted in the production of high levels of tryptamine. [Table 13]

[0204] Part 2 In follow-up experiments, tryptamine-producing strains were further genetically modified to increase titer. The strains were constructed according to Example 1, and the list of constructed strains is shown in Table 5 and Table 6. The strains were cultured according to Example 4, and quantified according to Example 8. The results shown in Table 13 and Figure 14 show that further genetic modification significantly increases tryptamine titer. The obtained highest producing strain serves as an ideal starting point for further genetic modification of Saccharomyces cerevisiae to produce substituted tryptamine derivatives. [Table 14]

[0205] Conclusion: Through genetic modifications including wild-type gene overexpression, gene deletion, and heterologous gene expression in Saccharomyces cerevisiae, the unnatural metabolite tryptamine can be produced in significantly greater quantities, far beyond those reported in the art.

[0206] Example 12 - De novo production of the substituted tryptamine derivative serotonin by genetically engineered strains of Saccharomyces cerevisiae Part 1 The novel production of serotonin in yeast was achieved by combining the gene encoding RgTdc, which converts L-tryptophan to tryptamine, with the genes encoding enzymes (OsT5H and FoCPR) that convert tryptamine to serotonin. The integration plasmid containing these genes was introduced into Saccharomyces cerevisiae strain BY4741 as previously described. The genetically modified yeast strain was cultured and extracellular metabolites were extracted as described in Examples 4 and 8. The production of serotonin was quantified by comparison with an authenticated analytical serotonin standard by UHPLC analysis. The culture results are shown in Table 14. Overexpression of these genes successfully led to the production of serotonin. [Table 15]

[0207] It has been found that yeast can be genetically modified to produce serotonin by converting tryptophan to tryptamine (by tryptophan decarboxylase) followed by direct hydroxylation at the 5-position of the indole ring by tryptamine hydroxylase and cytochrome P450 reductase.This is a major advance over known methods in the art (e.g., WO2013127915), which utilize a tetrahydrobiopterin-dependent tryptophan hydroxylase enzyme to convert tryptophan to 5-hydroxytryptophan, which is then decarboxylated by 5-hydroxytryptophan decarboxylase to convert to serotonin.Tetrahydrobiopterin is not naturally produced in most microorganisms, including yeast, which requires the supplementation of this expensive cofactor, or extensive genetic engineering efforts to genetically engineer microorganisms to produce new cofactors.As shown herein, direct conversion from tryptamine is not only more efficient, but also reduces the need to consider this additional cofactor. Also unexpected is the observation that CYP / CPR pairs from different organisms function efficiently in yeast. Typically, a cytochrome P450 (CYP) requires the aid of a specific cytochrome P450 reductase (CPR) to function. For example, many plant species have multiple CPR enzymes and are functional only with a specific CYP from that species. Interestingly, a CYP from rice (Oryza Sativa) (OsT5H) is functional with a CPR from Fusarium oxysporum (FoCPR).

[0208] Part 2 In a follow-up experiment, serotonin production was further improved by introducing OsT5H (SEQ ID NO: 95) and FoCPR (SEQ ID NO: 111) into a more tryptamine producing strain (SC-106). The strain was constructed according to Example 1, cultured according to Example 4, and quantified according to Example 8. The results shown in Table 15 and Figure 15 show a further improvement in serotonin titer with SC-75 producing significantly higher amounts of serotonin, indicating that the OsT5H / FoCPR enzyme pair has a large biocatalytic potential to convert tryptamine to serotonin. The results also show that expression of tryptamine 5-hydroxylase (OsT5H) alone is insufficient to enable efficient serotonin production, and furthermore, the concerted action of the CYP / CPR pair is necessary for full catalytic activity. Only trace amounts of serotonin were detected in strains expressing OsT5H without FoCPR (SC259), indicating the importance of efficient CPR co-expression. Unless S. cerevisiae contains a wild-type CPR enzyme (Ncp1), it does not normally function with heterologous CYP enzymes such as tryptamine 5-hydroxylase. Indeed, the results shown in Table 15 indicate that in SC-259, a strain expressing OsT5H but not a heterologous CPR (as per the wild-type yeast Ncp1), minimal production of serotonin is observed. [Table 16]

[0209] Conclusion: This experiment shows that Saccharomyces cerevisiae can be genetically modified to efficiently produce serotonin through an alternative biosynthetic pathway that was not known in the art. This production method is more efficient, but also provides a significant improvement over the prior art by mitigating the need to produce or exogenously add tetrahydrobiopterin. This experiment also shows that functional expression of tryptamine 5-hydroxylase requires the co-expression of a cytochrome P450 reductase, such as FoCPR, for full catalytic activity. Finally, this experiment shows the unexpected finding that FoCPR significantly enhances the activity of OsT5H, even though it is from a completely different organism.

[0210] Example 13 - De novo production of the substituted tryptamine derivative 4-coumaroylserotonin by genetically engineered Saccharomyces cerevisiae strains By combining the enzymes for the de novo production of tryptamine (RgTdc), the enzymes that derivatize tryptamine to serotonin (OsT5H, FoCPR), the enzymes for the de novo production of 4-coumaroyl-CoA (ARO7(G141S), ARO8, Pal2, C4h, Atr2, 4Cl) and finally the enzyme that derivatizes serotonin with 4-coumaroyl-CoA to produce 4-coumaroyl-serotonin (CaSHT), we realized the de novo production of the high-value molecule 4-coumaroyl-serotonin from safflower. The integrative plasmids encoding these genes were introduced into Saccharomyces cerevisiae as described above.

[0211] The genetically modified strains were cultured as described above, and the production of 4-coumaroylserotonin as well as other pathway intermediates was quantified by HPLC analysis by comparison with certified analytical standards. The genotypes of each strain tested are shown in Table 5, while the results of the culture experiments are shown in Table 16 and Figure 16. Overexpression of these genes successfully produced 4-coumaroylserotonin. [Table 17]

[0212] Conclusion: The results of this culture experiment show that yeast can be genetically modified to efficiently produce conjugated serotonin derivatives such as 4-coumaroylserotonin (4CS), a molecule with applications as a highly potent antioxidant, tyrosinase inhibitor, and anti-hyperpigmentation agent.

[0213] Example 14 - De novo production of other substituted tryptamine derivatives by genetically modified Saccharomyces cerevisiae strains By combining one or several derivatizing enzymes with a strain producing tryptamine, a series of substituted tryptamine derivatives can be produced. This was demonstrated by constructing a strain producing tryptamine (expressing RgTdc), then converting tryptamine to serotonin (by expressing OsT5H and FoCPR), which was then converted to more complex derivatives such as O-methylserotonin by expressing HsASMT, and N-methylserotonin by expressing CsSNMT1. The strains were cultured, extracted, and analyzed by LC-MS / QTOF as described previously to confirm the production of substituted tryptamine derivatives by the genetically engineered strains, as shown in Table 17. [Table 18]

[0214] Conclusion: The results of this experiment show that yeast strains genetically modified to produce serotonin can be further genetically modified to produce more highly conjugated serotonin derivatives. The results also surprisingly show that HsASMT, an enzyme said to convert normelatonin to melatonin, also has catalytic activity on serotonin and can convert it to O-methylserotonin. Finally, these results show that a putative methyltransferase from orange (Citrus sinensis) (CsSNMT1) is also active on serotonin.

[0215] Example 15 - Production of substituted tryptamine derivatives by feeding substituted indoles to genetically engineered Saccharomyces cerevisiae strains The production of 5-fluorotryptamine and 5-methoxytryptophan was achieved by feeding substituted indoles to genetically modified Saccharomyces cerevisiae strains constructed as described in Example 2. The strains contained a centromeric plasmid containing genes encoding PfTrpB (2B9) for the conversion of substituted indoles to substituted tryptophans and RgTdc for the conversion of substituted tryptophans to the corresponding substituted tryptamines. RgTdc was placed under the control of the GAL10 promoter and expression was induced with galactose after initial incubation with glucose. The yeast strains were cultured as described in Example 5 supplemented with 1 mM 5-fluoroindole or 1 mM 5-methoxyindole to produce 5-fluorotryptamine and 5-methoxytryptophan, respectively. Metabolites were extracted and analyzed by LC-MS / MS as previously described. The results of the LC-MS / MS analysis are shown in Table 18. It was found that strains expressing heterologous synthases could successfully convert substituted indoles to substituted tryptophans, but surprisingly, wild-type S. cerevisiae (BY4741) could also catalyze the reaction of successfully converting 5-methoxyindole to 5-methoxytryptophan, indicating that the wild-type S. cerevisiae enzyme Trp5 also has the ability to convert substituted indoles to their corresponding substituted tryptophans. It was also found that tryptophan decarboxylase, in combination with heterologous tryptophan synthase and tryptophan decarboxylase, could convert substituted tryptophans to their corresponding substituted tryptamines, as shown by the conversion of 5-fluoroindole to 5-fluorotryptamine. [Table 19]

[0216] Conclusion: This experiment shows that in the absence of de novo production of the desired substituted tryptamine derivatives, yeast can also similarly produce these compounds by introducing substituted indole precursors supplied in the culture medium, and can produce and export the corresponding substituted tryptophan and / or tryptamine derivatives using heterologous tryptophan synthases and tryptophan decarboxylases with broad substrate scope.

[0217] Example 16 - Production of Iboga Alkaloids by Bioconversion Using Genetically Engineered Saccharomyces cerevisiae Iboga alkaloids, including ibogaine and noribogaine, are a therapeutically relevant class of molecules that have recently been investigated as potential treatments for addiction. Selection of raw material sources for these molecules is currently difficult due to the scarcity of the wild-type species that produces these molecules, iboga (Tabanante iboga), as well as the relatively low abundance of the molecules in these hosts. Alternatively, genetically modified yeast can be used to convert precursor molecules from more abundant sources into the desired final iboga alkaloids.

[0218] As an example, the production of ibogaine and noribogaine was achieved by biotransformation of alkaloids found in the abundant root bark of Tabernaemontana alba. Extraction and decarboxylation were performed according to (Krengel et al., 2019) to obtain a methanol extract of the alkaloids lacking the methoxy moiety.

[0219] 40 ml of methanol was added to 50 ml Falcon tubes containing 3 g of dried, powdered root bark from each species and allowed to soak or be sonicated for 60 min. During this time, the tubes were vortexed 4 times and finally centrifuged at 2400 rpm for 5 min. The supernatant was collected by pipetting through cotton wool and evaporated to dryness.

[0220] 450 mg of each extract was dissolved separately in 30 ml of a mixture of methanol and potassium hydroxide (MeOH / KOH mixture was made by dissolving 9 g of KOH in 2 ml of water and diluted with methanol to a final volume of 60 ml) in 50 ml Pyrex tubes, which were then immersed in a hot water bath and maintained at an internal temperature of 72-75 °C for 2 hours. The saponified methanol extract was evaporated to dryness. The latter was suspended separately in 25 ml of 2 M aqueous hydrochloric acid and gravity filtered through Whatman grade 2 filter paper. The filtrate was heated to 95 °C in a 50 ml Pyrex tube and immersed in a hot water bath. After 15 minutes, the solution was cooled at room temperature before the pH was raised to 10 by ammonium hydroxide and gravity filtration. The resulting filtrate and dried precipitated material were extracted five times each with dichloromethane, which after evaporation gave the saponified and decarboxylated alkaloid extract.

[0221] The demethoxycarbonylated alkaloid extract was dissolved in ethanol and fed to a genetically modified Saccharomyces cerevisiae strain containing enzymes from the ibogaine biosynthetic pathway (final ethanol concentration of 2%). The cells expressed Tabernanthe iboga ibogamine-10-hydroxylase (PL-533 (Rec 2-LEU:TiI10H), SEQ ID NO: 99) and cytochrome P450 reductase (PL-535 (Rec 3x-XI-5:CrCPR1) or PL-536 (Rec 3x-XI-5:CrCPR2), SEQ ID NO: 107 or 109), either alone or in combination with iboganoribogaine 10-O-methyltransferase (PL-534 (Rec 1-XI-5-LEU:TiN10OMT), SEQ ID NO: 119). Cultures were incubated in 500 μL of Delft medium with the appropriate amino acids added for 72 h at 30 °C. Whole cell broths were centrifuged at 4000 x g for 5 min and the supernatants were extracted and analyzed by UHPLC or LC-MS / MS. Whenever possible, authenticated analytical standards were used to confirm the identity of the produced molecules.

[0222] Example 17 - Construction of E. coli strains for the production of substituted tryptamine glycosides derived from substituted tryptamine An E. coli strain expressing a gene that converts substituted tryptamine to substituted tryptamine glycoside was constructed as follows. The glycosyltransferase gene was codon-optimized for E. coli, synthesized by Twist, and cloned into a custom plasmid vector (pRSGLY, synthesized by GeneArt) using standard restriction ligation with SpeI / XhoI restriction enzyme sites. This custom vector contained a LacI operon, an AmpR cassette, an origin of replication, and multiple cloning sites flanked by a T7 promoter and terminator. In addition, the 5' end also contained a ribozyme binding site (RBS) and a 6xHis tag for subsequent protein purification. The fully assembled plasmid was transformed into E. coli DH5α or E. coli XJb(DE3) autolytic strain (Zymo Research). Plasmids encoding substituted tryptamine glycosyltransferase are shown in Table 19. In some cases, when E. coli strains were used to produce and purify enzymes for in vitro reactions, the glycosyltransferases were expressed on themselves to facilitate substituted tryptamine feeding experiments, as well as to facilitate in vitro biocatalysis experiments. [Table 20]

[0223] Example 18 - In vitro testing of the performance of glycosyltransferases in the glycosylation of substituted tryptamines For in vitro testing of the performance of glycosyltransferases in attaching glucose moieties onto the free hydroxy groups of various substituted tryptamines, purified glycosyltransferases were prepared as described in Example 7, enzyme assays were performed as described in Example 10, and quantification of glucoside production was performed as described in Example 8. Alternatively, when quantification by analysis of peak areas by HPLC was not possible due to poor separation of the substituted tryptamine and its corresponding glucosides, characterization was performed by LC-MS / QTOF where the relative peak areas of both the glucosides and substrate obtained from LC-MS / QTOF were used to calculate the % conversion of the substituted tryptamine to its corresponding glucoside.

[0224] In the initial screen, glucosylation was tested with the substituted tryptamines serotonin, psilocin, bufotenin and noribogaine using UDP-glucose as the sugar donor. The corresponding structure ID (OBT-001-OBT-004) was given for each substituted tryptamine glucoside produced in this screen, and the structure and trivial name of each molecule are shown in Figure 4. Figure 4. Structures of substituted tryptamine glucosides verified by LC-MS / QTOF.

[0225] Substituted tryptamine glucosides produced using psilocin as an acceptor. A number of glycosyltransferases were found to catalyze the conversion of psilocin to OBT-001 (psilocin-O-β-D-glucoside) (FIG. 4). FIG. 5 shows representative chromatograms generated by LC-MS / QTOF analysis of reaction mixtures containing substituted tryptamines and exemplary glycosyltransferase At71C2 (SEQ ID NOs: 193, 194). The figure further shows the retention time (RT), predicted and measured mass of each compound, as well as the fragmentation patterns determined by LC-MS / QTOF analysis and thereby confirming the structures of the produced glucosides.

[0226] It was further revealed that multiple glycosyltransferases could catalyze the conversion of psilocin to OBT-001 (psilocin-O-β-D-glucoside) with various conversion efficiencies. Table 20 shows the glycosyltransferases that produced psilocin-O-β-D-glucoside with the % conversion efficiency. [Table 21]

[0227] Substituted tryptamine glucosides produced using noribogaine as an acceptor. A number of glycosyltransferases were found to catalyze the conversion of noribogaine to OBT-002 (noribogaine-O-β-D-glucoside) (FIG. 4). FIG. 6 shows representative chromatograms generated by LC-MS / QTOF analysis of reaction mixtures containing substituted tryptamines and exemplary glycosyltransferases. The figure further shows the retention time (RT), predicted and measured mass of each compound, as well as the fragmentation patterns determined by LC-MS / QTOF analysis and thereby confirming the structures of the product glycosides.

[0228] It was further revealed that multiple glycosyltransferases could catalyze the conversion of noribogaine to OBT-002 (noribogaine-O-β-D-glucoside) with various conversion efficiencies. Table 21 shows the glycosyltransferases that produced noribogaine-O-β-D-glucoside along with the % conversion efficiency. [Table 22]

[0229] Substituted tryptamine glucosides produced using bufotenin as an acceptor. A number of glycosyltransferases were found to catalyze the conversion of bufotenin to OBT-003 (bufotenin-O-β-D-glucoside) (FIG. 4). FIG. 7 shows representative chromatograms generated by LC-MS / QTOF analysis of reaction mixtures containing substituted tryptamines and exemplary glycosyltransferases. The figure further shows the retention time (RT), predicted and measured mass of each compound, as well as the fragmentation patterns determined by LC-MS / QTOF analysis and thereby confirming the structures of the product glucosides.

[0230] It was further revealed that multiple glycosyltransferases could catalyze the conversion of bufotenin to OBT-003 (bufotenin-O-β-D-glucoside) with various conversion efficiencies. Table 22 shows the glycosyltransferases that produced bufotenin-O-β-D-glucoside with the % conversion efficiency. [Table 23]

[0231] Substituted tryptamine glucosides produced using serotonin as a receptor. A number of glycosyltransferases were found to catalyze the conversion of serotonin to OBT-004 (serotonin-O-β-D-glucoside) (FIG. 4). FIG. 8 shows representative chromatograms generated by LC-MS / QTOF analysis of reaction mixtures containing substituted tryptamines and exemplary glycosyltransferases. The figure further shows the retention time (RT), predicted and measured mass of each compound, as well as the fragmentation patterns determined by LC-MS / QTOF analysis and thereby confirming the structures of the product glycosides.

[0232] It was further revealed that multiple glycosyltransferases could catalyze the conversion of serotonin to OBT-004 (serotonin-O-β-D-glucoside) with various conversion efficiencies. Table 23 shows the glycosyltransferases that produced serotonin-O-β-D-glucoside with the % conversion efficiency. [Table 24]

[0233] Overall, it was found that a series of glycosyltransferases can use various substituted tryptamines as sugar acceptors to produce a series of novel substituted tryptamine glycosides. In the screening, enzymes capable of catalyzing a wide variety of highly specific reactions were found. It was found that glycosyltransferases can selectively attach a single glucose group onto a substituted tryptamine molecule to produce the corresponding monoglycoside, e.g., psilocin-O-β-D-glucoside (OBT-001) by Sp72T (SEQ ID NO: 191, 192). Based on the calculated conversion percentage, it was found that many glycosyltransferases are highly active and can utilize UDP glucose to efficiently glucosylate substituted tryptamines with high conversion efficiency. For example, At71C1-Sr71E1_354 (SEQ ID NOs: 199, 200) was found to efficiently produce glucosides of psilocin, bufotenin, and serotonin, while Pt73Y (SEQ ID NOs: 203, 204) was found to efficiently produce noribogaine glucoside. A large number of enzymes were found to catalyze glycosylation reactions on substituted tryptamines, and in total, this in vitro screen revealed that 30 glycosyltransferases were identified that were active on substituted tryptamine molecules.

[0234] Conclusion: The results of this experiment show that UGT enzymes from a diverse set of plant species surprisingly catalyze the glycosylation of substrates not encountered in the natural environment of their host species, leading to the production of novel tryptamine glycosides. The experiment also revealed surprisingly high catalytic activity of several UGTs tested, including enzymes such as At71C2, At71C1_At71C2_353, and Pt73Y, which convert substantial amounts of available substrates to their corresponding glycosides.

[0235] Example 19 - In vitro testing of glycosyltransferase performance in glucosylating substituted tryptamines with alternative UDP sugars In Example 18, glycosyltransferases were found that could accept a range of UDP glucoses and catalyze the production of a range of substituted tryptamine glucosides. The most highly functional enzymes from this screen were further tested to determine whether they could accept another UDP sugar and catalyze the production of substituted tryptamine glycosides with different sugar groups attached. For in vitro testing of the performance of glycosyltransferases in the glycosylation of substituted tryptamines, purified glycosyltransferases were prepared as described in Example 7, enzyme assays were performed as described in Example 10, and quantification of glucoside production was performed as described in Example 8. Alternatively, when quantification by analysis of peak areas by HPLC was not possible due to poor separation of the substituted tryptamines and their corresponding glucosides, quantification was performed using LC-MS / QTOF as described in Example 18.

[0236] In the initial screen, glycosylation was tested using UDP-xylose as the sugar donor. Xylosylation of noribogaine and bufotenin was tested with Pt73Y (SEQ ID NO: 203, 204), and xylosylation of psilocin with At71C2 (SEQ ID NO: 193, 194). The corresponding structure ID is given for each substituted tryptamine glucoside produced in this screen, and the structure of each molecule is shown in FIG. 9. The resulting LC-MS / QTOF chromatograms produced by each reaction are shown in FIG. 10 for OBT-005, FIG. 11 for OBT-006, and FIG. 12 for OBT-007.

[0237] It was found that each test enzyme could efficiently xylosylate their respective substrates producing the resulting xylosides. Calculation of the percent conversion revealed that Pt73Y (SEQ ID NOs: 203, 204) converted 15% of the available noribogaine and 7.9% of the available bufotenine to their respective xylosides, while At71C2 (SEQ ID NOs: 193, 194) converted 8.2% of the available psilocin to its respective xylosides. These results indicate that the glycosyltransferase enzymes discovered can bind not only glucose, but other sugars as well, resulting in a more diverse range of substituted tryptamine glycosides.

[0238] Conclusion: The results of this experiment show that these UGTs not only have a broad substrate range in terms of the aglycone substrate, but they also have a broad substrate range for the UDP sugars used: not only do these UGTs accept UDP-glucose as a substrate, they can accept other sugars such as UDP-xylose as well.

[0239] Example 21 - Improved psilocybin production in genetically modified Saccharomyces cerevisiae strains by deletion of the wild-type phosphatase gene It was observed that S. cerevisiae strains genetically modified to produce psilocybin by the integration of heterologous psilocybin biosynthetic pathway genes accumulated significant concentrations of psilocin (e.g., Table 34). It is assumed that this accumulation was due to cleavage of phosphate groups. It was further decided to test whether the degradation of psilocybin to psilocin was due to enzymatic hydrolysis by endogenous S. cerevisiae phosphatase enzymes. It was found that the psilocybin phosphatase from Psilocybe cubensis (PcPsiP) shares significant sequence homology with several wild-type S. cerevisiae phosphatases. In Psilocybe cubensis, the wild-type function of PcPsiP was found to convert psilocybin to psilocin, thus further revealing that the wild-type S. cerevisiae enzymes share significant sequence homology and that they also have promiscuous activity against psilocybin and convert it to psilocin as well. To test this hypothesis, several Saccharomyces cerevisiae phosphatase genes were knocked out in the psilocybin-producing strain SC-276 (according to Example 1), and the effect on the amount of produced psilocybin and psilocin was measured from standard cultures (according to Example 4), quantified using standard HPLC analysis, and compared to certified analytical standards (according to Example 8). The results shown in Table 24 and Figure 17 show a large improvement in psilocybin titer in several knockout strains. In general, knocking out these phosphatase genes results in significantly higher psilocybin production. In particular, knocking out DIA3, PHO5, and PHO3 produces the greatest increase in psilocybin. [Table 25]

[0240] Conclusion: The results of this experiment indicate that the high psilocybin degradation observed in the genetically modified yeast strains with homology to PsiP from D. cerevisiae was due to dephosphorylation by wild-type S. cerevisiae phosphatase enzymes. Deletion of these phosphatase genes resulted in a significant improvement in psilocybin production due to a marked reduction in the degradation of psilocybin to psilocin. This further demonstrated that deletion of one or more of these genes was a successful strategy to improve production.

[0241] Example 22 - In vitro production of substituted tryptamine derivatives by one-pot biocatalytic enzyme cascade using serine and substituted indole derivatives Part 1 In Example 15, production of the substituted tryptamine derivatives 5-fluorotryptamine and 5-methoxytryptophan was achieved in culture experiments by Saccharomyces cerevisiae strains overexpressing the genetically engineered tryptophan synthase PfTrpB (2B9) (and tryptophan decarboxylase RgTdc in the case of 5-fluorotryptamine) and feeding the respective substituted indoles (5-fluoroindole, 5-methoxyindole). In this experiment, the wild-type, non-genetically modified tryptophan synthase enzyme from Dactyla cruciata (PcTrpB, SEQ ID NO: 179) was compared to genetically modified tryptophan synthases known in the art (PfTrpB(2B9), SEQ ID NO: 59 and TmTrpB(M145T, N167D), SEQ ID NO: 67). A PcTrpB enzyme containing the equivalent mutations as TmTrpB(M145T, N167D) was also generated for use as a comparison (PcTrpB(M439T, N459D), SEQ ID NO: 181). The gene was cloned into a pET28a(+) expression plasmid and cloned into E. coli according to Example 3. Purified enzyme was prepared according to Example 7. In the wild-type reaction, tryptophan synthase converts indole and serine to tryptophan, and in this assay we investigated whether different tryptophan synthases can accept different substituted indoles and serines as substrates for the corresponding substituted tryptophans. In vitro enzyme reactions were set up according to Table 25 and incubated at 30° C. for 16 hours. [Table 26]

[0242] After 16 hours, the reaction was stopped by freezing. Samples were analyzed by HPLC according to Example 8. The amount of substituted indole converted to its corresponding substituted tryptophan (%) was calculated by peak area and the assay results are shown in Table 26. [Table 27]

[0243] The results showed that the tryptophan synthase enzymes tested accepted a wide range of substituted indole derivatives and produced the corresponding substituted tryptophan derivatives. As expected, genetically modified tryptophan synthase enzymes with amino acid modifications known in the art that substantially increased the substrate scope of these enzymes had great activity with a wide range of substituted indole derivatives, but surprisingly, the wild-type tryptophan synthase from PcTrpB had activity comparable to, and in many cases superior to, its genetically modified counterpart. PcTrpB and the genetically engineered version of PcTrpB (M439T, N459D) outperformed both TmTrpB (M145T, N167D) and PfTrpB (2B9), both of which are known in the art to have better catalytic activity than wild-type tryptophan synthase with virtually all substituted indoles tested, while wild-type PcTrpB was even better than its genetically engineered counterpart PcTrpB (M439T, N459D) with many substrates, including 4-hydroxyindole, 5-methoxyindole, 5-nitroindole, and 7-nitroindole. No activity was detected using azaindole as a substrate, likely due to the low solubility of the substrate. The results surprisingly clearly show the broad substrate scope of wild-type tryptophan synthase (PcTrpB). Although the prior art has reported extensive genetic modification and enzyme evolution efforts to increase the substrate scope and catalytic activity of the tryptophan synthase enzyme, this study shows that the non-genetically modified enzyme from D. cerevisiae significantly outperforms these genetically modified enzymes, paving the way for its use as a superior enzyme catalyst for the production of a diverse range of substituted tryptophan derivatives.

[0244] Part 2 To further demonstrate the usefulness of fungal enzymes, a coupled reaction was constructed using tryptophan synthase (PcTrpB) and a series of tryptophan decarboxylases. Although tryptophan is the natural substrate of tryptophan decarboxylase, which catalyzes the formation of the corresponding amine (tryptamine), the prior art has reported enzymes that exhibit a broader substrate scope and the ability to produce a series of substituted tryptamines from the corresponding substituted tryptophan derivatives. In particular, it is known in the art that RgTdc (SEQ ID NO: 69) is particularly effective when combined with genetically engineered tryptophan synthase (TmTrpB (M145T, N167D)) and PfTrpB (2B9) in a one-pot reaction to produce substituted tryptamine derivatives from the corresponding substituted indole derivatives and serine. RgTdc was used as a benchmark to compare the activity and substrate scope of two fungal tryptophan decarboxylases from D. cerevisiae (PcPsiD, SEQ ID NO: 71) and the related psilocybin-producing fungus Panaeolus cyanescens (PanCyPsiD, SEQ ID NO: 77), as well as a non-standard amino acid decarboxylase from D. cerevisiae (PcncAAD, SEQ ID NO: 73). In this assay, the ability of different tryptophan decarboxylases to convert substituted tryptophan derivatives produced by the conversion of substituted indoles and serines by PcTrpB was investigated. Purified enzymes were prepared according to Example 7, and enzyme assays were configured according to Table 27 below and incubated at 30°C for 16 hours. [Table 28]

[0245] After 16 hours, the reaction was stopped by freezing. Samples were analyzed by HPLC according to Example 8. The amount of substituted indole converted to its corresponding substituted tryptamine (%) was calculated by peak area and the assay results are shown in Table 28. [Table 29]

[0246] The results showed that PcncAAD had negligible activity toward the substituted tryptophan derivatives produced by PcTrpB (probably because indole molecules are generally outside its substrate scope), whereas the other three tryptophan decarboxylases had considerable activity. Not surprisingly, RgTdc, known in the art to have a large substrate scope toward a series of substituted tryptophan derivatives, had good activity toward most tested substrates, but the two fungal tryptophan decarboxylases were better or equal to RgTdc toward almost all tested substrates, with only 6-bromoindole having higher activity than RgTdc. Interestingly, RgTdc had no detectable activity toward 7-bromoindole, whereas both fungal enzymes could efficiently convert it to 7-bromotryptamine with a conversion rate of 75%. Again, the results clearly demonstrate the broad substrate scope of the wild-type enzymes from D. cerevisiae and D. nigricans and their potential to efficiently produce a wide range of substituted tryptamine derivatives.

[0247] These fungal tryptophan synthase and tryptophan decarboxylase combinations allow for the efficient one-pot production of a wide range of substituted tryptamines from their corresponding substituted indoles and serines. From an industrial context, this is important because substituted tryptamines are typically difficult to produce and often very expensive, whereas substituted indoles, in contrast, are relatively easy to produce and often very inexpensive.

[0248] Conclusion: The results of this experiment showed that the wild-type tryptophan synthase from D. cerevisiae has a fairly broad substrate range and can accept a wide range of substituted indoles to produce the corresponding substituted tryptophans. This is a surprising finding that overcomes the preconception in the art that wild-type tryptophan synthase enzymes do not have a broad substrate range unless genetically modified with "activating mutations" corresponding to residues 144 and 166, or residue 292 (e.g., US Patent Publication No. 20160298152A1). These results showed that, indeed, some tryptophan synthase enzymes, such as TmTrpB and PfTrpB, required these activating mutations to expand their substrate range, but not all tryptophan synthases do, and in fact, the wild-type tryptophan synthase from D. cerevisiae described herein is more efficient and has a broader substrate range than genetically modified enzymes reported in the art. Furthermore, these results also showed that the introduction of the corresponding activating mutations into the wild-type tryptophan synthase PcTrpB did not improve catalytic activity and substrate scope, and in fact, in some cases, resulted in lower activity.Finally, these results showed that tryptophan decarboxylases from D. cerevisiae and D. nigricans (PcPsiD and PanCyPsiD) also surprisingly have a broad substrate scope, at least comparable to, or in some cases superior to, the promiscuous tryptophan decarboxylases (RgTdc) known in the art.Altogether, this indicates that psilocybin-producing mushroom species are an underutilized source of useful enzymes with exciting new bioindustrial applications that offer advantages over methods known in the art.

[0249] Example 23 - Combination of additional derivatizing enzymes to the tryptophan synthase / tryptophan decarboxylase one-pot biocatalytic enzyme cascade with serine and substituted indole derivatives leads to more highly complex substituted tryptamine derivatives In Example 22, a biocatalytic cascade was demonstrated for the production of diverse substituted tryptamine derivatives from serine and substituted indole derivatives using a series of tryptophan synthase and tryptophan decarboxylase enzymes. To further demonstrate the utility of this one-pot cascade, a reaction was constructed that included wild-type tryptophan synthase and tryptophan decarboxylase from Pseudomonas niger (PanCyTrpB, SEQ ID NO: 255 and PanCyPsiD, SEQ ID NO: 77) in combination with additional enzymes that could further convert the substituted tryptamine derivatives produced by PanCyTrpB and PanCyPsiD to more complex derivatives when fed with the corresponding substituted indole derivatives and serine. In this example, PanCyPsiK (SEQ ID NO: 159) and PanCyPsiM (SEQ ID NO: 127) were added as additional derivatizing enzymes, and serine and 4-hydroxyindole were used as the feeding substrates. The promiscuous PanCyTrpB and PanCyPsiD converted these substrates to 4-hydroxytryptamine, which was further derivatized by PanCyPsiK to norbaeocystin, followed by PanCyPsiM-mediated derivatization to baeocystin and then to psilocybin. The assay was configured according to Example 22 with 6 mM serine and 3 mM 4-hydroxyindole, and the results of the experiment are shown in Table 29. The results show that psilocybin can be efficiently produced from serine and 4-hydroxyindole using a one-pot biocatalytic cascade. Decomposition to psilocin was also observed due to the instability of the phosphate group of psilocybin. [Table 30]

[0250] Conclusion: These results indicate that the excellent catalytic activities of PanCyTrpB and PanCyPsiD can be further utilized as starting points for producing more highly complex substituted tryptamine derivatives.

[0251] Example 24 - Production of N-feruloyl serotonin by genetically modified Saccharomyces cerevisiae In Example 13, yeast was genetically modified to produce the highly potent antioxidant and anti-hyperpigmentation compound 4-coumaroylserotonin by introducing genes for the production of serotonin from tryptophan (RgTdc, OsT5H, FoCPR), genes for the production of 4-coumaroyl-CoA from phenylalanine (ARO7(G141S), ARO8, Pal2, C4h, Atr2, 4Cl), and finally an enzyme (CaSHT) that derivatizes serotonin with 4-coumaroyl-CoA to produce 4-coumaroylserotonin. Since N-(hydroxycinnamoyl)transferase (CaSHT) is known to functionalize serotonin, a series of hydroxycinnamoyl derivatives including coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA and feruloyl-CoA leading to the production of N-coumaroylserotonin (described above), N-caffeoyl-serotonin, N-cinnamoyl-serotonin and N-feruloyl-serotonin, functional expression of CaSHT opened the possibility of producing other serotonin derivatives. To further demonstrate the utility of this enzyme, yeast was genetically modified to produce N-feruloyl serotonin from exogenously supplied ferulic acid by introducing genes that produce serotonin from tryptophan (CrTdc, SEQ ID NO: 25; OsT5H, SEQ ID NO: 95; FoCPR, SEQ ID NO: 111), a gene that converts ferulic acid to feruloyl CoA (4CL2, SEQ ID NO: 57), and a gene that conjugates feruloyl CoA and serotonin to N-feruloyl serotonin (CaSHT, SEQ ID NO: 161). A strain (SC-NFS) was constructed according to Example 1, cultured with 1 mM ferulic acid added to the medium according to Example 4, and the concentration of N-feruloyl serotonin was quantified according to Example 8. As shown in Table 30, SC-NFS was able to convert significant amounts of exogenously added ferulic acid to N-feruloyl serotonin, thereby demonstrating the utility of the CaSHT enzyme and the use of yeast to produce conjugated serotonin derivatives. [Table 31]

[0252] Conclusion: These results indicate that hydroxycinnamoyl derivatives such as N-feruloylserotonin can also be easily produced by supplying an inexpensive hydroxycinnamic acid (in this case, ferulic acid) rather than producing the corresponding cinnamic acid de novo (as shown for the de novo production of 4-coumaroylserotonin).

[0253] Example 25 - Melatonin production by genetically modified yeast strains In Example 12, it was shown that serotonin can be produced in yeast by integrating and expressing tryptophan decarboxylase (e.g., CrTdc) and CYP / CPR pair, tryptamine 5-hydroxylase and P450 reductase (OsT5H, FoCPR). Examples 13 and 24 further showed how this serotonin producing strain can be further genetically modified to produce more complex derivatives (4-coumaroylserotonin, N-feruloylserotonin). Here, the serotonin producing strain (SC-75) was further genetically modified to produce human hormone and dietary supplement melatonin by further integrating and expressing acetylserotonin methyltransferase (HsASMT, SEQ ID NO: 117) and serotonin N-acetyltransferase (BtAANAT, SEQ ID NO: 141). The strain was constructed according to Example 1, cultured according to Example 4, and quantified according to Example 8. As shown in Table 31 and FIG. 19, integration of serotonin producing strains of HsASMT and BtAANAT produced melatonin. [Table 32]

[0254] Conclusion: The results of this experiment indicate that efficient serotonin production by OsT5H and FoCPR can be further utilized to produce more highly complex serotonin derivatives such as melatonin, which provides a significant improvement over microbial methods for producing melatonin known in the art (e.g., WO2013127915) that rely on an inefficient 5-hydroxytryptophan-mediated serotonin production pathway that requires a tetrahydrobiopterin cofactor.

[0255] Example 26 - Production and purification of psilocin-O-β-glucoside by one-pot in vitro biocatalytic cascade using 4-hydroxyindole, serine and UDP-glucose For further testing, in order to produce more psilocybin glucoside, a one-pot in vitro biocatalytic cascade was constructed to produce psilocybin glucoside directly from 4-hydroxyindole, serine and UDP glucose. In a similar manner to Example 23, PanCyTrpB, PanCyPsiD, PanCyPsiK and PanCyPsiM were added to construct a reaction that converts 4-hydroxyindole and serine to psilocybin. In Example 23, the degradation of psilocybin to psilocin was observed, where we utilized it to provide a substrate for UGT enzyme At71C2 (SEQ ID NO:193, 194), which was also added to the one-pot reaction with UDP glucose, and the available psilocin was converted to psilocin-O-β-glucoside as psilocybin was degraded from psilocybin. The reaction was scaled up to 20 mL (as in Example 10) by adding 15 mg of each enzyme to a reaction containing 1 mM 4-hydroxyindole, 1 mM serine, and 3 mM UDP-glucose, as well as alkaline phosphatase. The reaction was incubated at 30° C. for 24 hours, then terminated by heating at 80° C. to degrade the protein and any unreacted psilocybin and psilocin, and then filtered through a 0.2 μm polyvinylidene fluoride (PVDF) filter prior to purification by preparative HPLC. This resulted in a final psilocin-O-β-glucoside concentration of 0.3 mg / mL, representing a yield of approximately 75% from 4-hydroxyindole and serine. Psilocin-O-β-glucoside was purified from the reaction mixture by preparative HPLC as follows: The filtered assay mixture was loaded onto a Phenomenex Kinetex F5 column (250x21.2mm, 5um, 100Å) on an Agilent 1290 preparative HPLC equipped with a fraction collector and UV detector. Gradient elution was employed with a flow rate of 15mL / min using water with 0.01% trifluoroacetic acid (TFA) and methanol with 0.01% TFA as mobile phases A and B, respectively. The gradient was as follows: 0-1 min: 2% B, 1-30 min: 2-98% B, 30-35 min: 98% B, 30-37 min: 98-2% B.Peaks with areas greater than 15 units and ascending slopes greater than 0.60 units / sec were collected using automated peak detection at 230 nm. Fractions containing psilocin glucoside were dried under reduced pressure to give a white powder (final yield: 7.9 mg). The identity of psilocin-O-β-glucoside was confirmed by LC-MS / QTOF: calcd [M+H]. + :m / z 367.1864. Actual measurement [M+H] + : m / z 367.1865. MS2(367.1865): m / z 205.1339 corresponds to loss of the glucoside moiety.

[0256] Example 27 - Chemical stability test of psilocybin glucoside Prior art has shown that the psychotomimetic compound psilocin is highly unstable and degrades quickly even under ambient conditions. Although the additional phosphate groups of psilocybin help protect the molecule from degradation, psilocybin is also relatively unstable and prone to dephosphorylation to psilocin and subsequent degradation. The instability of these molecules hampers the formulation and delivery of the compounds for therapeutic use. Glycosylation can be used as an effective strategy to improve the chemical stability of the active compound psilocin. To demonstrate the effectiveness of psilocin glycosylation, purified psilocin glucoside was exposed to a series of harsh environmental conditions along with psilocin and psilocybin according to Example 9. The degradation of each compound was evaluated by measuring the amount of compound remaining after 24 hours of exposure to each condition. As shown in Table 32, psilocin-O-β-glucoside exhibited remarkable stability in all conditions tested, and showed virtually no signs of degradation even under particularly harsh conditions. In stark contrast, psilocin showed complete degradation under all conditions tested, with zero percent psilocin detected after 24 hours of exposure, thereby demonstrating the high instability of the compound. Psilocybin showed much less degradation compared to psilocin, and while the added phosphate group was shown to protect the molecule from degradation, it still showed signs of degradation and fell far short of psilocin glucoside in this stability study. These results demonstrate how glycosylation is a better strategy than phosphorylation to protect psilocin from degradation. [Table 33]

[0257] Conclusion: Psilocybin is a prodrug of psilocin, whereby the added phosphate group acts to stabilize the molecule and prevent degradation. This experiment shows that glycosylation of psilocin is a better strategy than phosphorylation (psilocybin) to stabilize the molecule, thereby providing an opportunity to develop novel prodrugs of this active molecule.

[0258] Example 28 - Production of psilocybin glucoside by genetically modified yeast In the previous examples, psilocin-O-β-glucoside was produced in vitro using the highly active UGT At71C2 (SEQ ID NO: 193), which converted psilocin to psilocin glucoside upon the addition of exogenous UDP-glucose. Psilocin glucoside was produced by feeding psilocin directly into a UGT reaction or by producing it in a one-pot biocatalytic cascade from 4-hydroxyindole and serine, relying on the instability of the intrinsic psilocybin to degrade to psilocin to provide the psilocin substrate for the glycosylation reaction. In this example, psilocybin glucoside was produced in yeast by two different production methods: a bioconversion method, whereby At71C2 was introduced into a wild type yeast strain (BY4741) and psilocybin glucoside was produced by feeding the strain with psilocin during cultivation, and a novel production method, whereby At71C2 was introduced into a yeast strain genetically modified to efficiently produce psilocybin (SC-276). The strain was constructed according to Example 1, cultivated according to Example 4, and quantified according to Example 8. For bioconversion cultivation, the strain was fed with 100 mg / L of psilocin. As shown in Table 33, the expression of At71C2 resulted in the production of psilocybin glucoside by both production methods. SC-276 produces psilocybin with psilocin produced as a by-product, but when At71C2 is introduced, (SC-402) psilocin is further converted to psilocybin glucoside. For the biotransformation strain (SC-394), only psilocin glucoside was detected, even when 100 mg / L of psilocin was supplied during the culture. The absence of additional psilocin is likely due to the highly unstable molecule. [Table 34]

[0259] Conclusion: This experiment demonstrated another method to produce tryptamine glycoside. While the previous example demonstrated tryptamine glycoside production using an in vitro biocatalytic approach, this experiment demonstrated that in vivo bioconversion and de novo production in yeast is also feasible. This opens multiple avenues for the industrial production of such novel molecules.

[0260] Example 29 - Production of psilocybin by genetically modified yeast expressing genes from Psilocybe punctata Part 1 Although D. fasciatus is probably the best known psilocybin-producing species, many species are known to produce psilocybin. However, to date, only the psilocybin biosynthetic pathway from D. fasciatus has been elucidated and the genes that convert tryptophan to psilocybin in five enzymatic steps have been discovered. Excavation of the genomes of other psilocybin-producing species could reveal novel gene homologs that may have improved activity compared to the well-known gene candidates from D. fasciatus. Genome mining identified a putative set of psilocybin biosynthetic genes in the fungal species P. cerevisiae, including a putative tryptophan decarboxylase (PanCyPsiD, SEQ ID NO:77), tryptamine 4-hydroxylase (PanCyPsiH, SEQ ID NO:87), cytochrome P450 reductase (PanCyCPR, SEQ ID NO:105), 4-hydroxytryptamine kinase (PanCyPsiK, SEQ ID NO:159), and psilocybin synthase (PanCyPsiM, SEQ ID NO:127). Interestingly, these putative genes share only about 80% amino acid sequence identity to their homologs from P. cerevisiae, and may exhibit distinct activities. To test the function of these enzymes and compare their activity against the homologous enzymes from P. cerevisiae, each gene set was introduced into a high tryptamine producing background (SC-106) according to Example 1, cultivated according to Example 4, and quantified for psilocybin and other by-products and intermediates according to Example 8 to directly compare the biocatalytic capabilities of the enzymes from each set. The results shown in Table 34 and Figure 18 indicate that introduction of genes from both species resulted in the production of psilocybin, but introduction of genes from P. cerevisiae resulted in greater production than genes from P. cerevisiae. Overall, the results indicate superior catalytic activity of the enzymes from P. cerevisiae. Less accumulation of tryptamine was also observed in SC-268 compared to SC-275, again supporting the improved activity of the P. cerevisiae biosynthetic pathway. [Table 35]

[0261] Part 2 Further genome mining identified a gene encoding a putative cytochrome b5 (PanCyCYB5, SEQ ID NO: 253). Heterologous expression of cytochrome b5 protein has previously been shown to improve CYP / CPR reactions (WO2021052989), but is usually specific to the expression of cytochrome b5 from the same species as the CYP / CPR pair, and therefore requires expression of P. cerevisiae cytochrome b5 to improve the activity of the PanCyPsiH / PanCyCPR CYP / CPR pair. The putative cytochrome b5 (PanCyCYB5) was integrated and expressed in a strain expressing the psilocybin biosynthetic pathway from P. cerevisiae according to Example 1, cultured according to Example 4, and the concentrations of psilocybin and other by-products and intermediates quantified according to Example 8. The results shown in Table 35 indicate that the additional expression of PanCyCYB5 improves psilocybin production by improving the conversion of tryptamine to 4-hydroxytryptamine. Some tryptamine accumulation was observed in SC-269, but no tryptamine was detected in SC-270, indicating improved hydroxylation activity and complete conversion of available tryptamine to 4-hydroxytryptamine. [Table 36]

[0262] Conclusion: This experiment shows that enzymes from other psilocybin species can be introduced into yeast, allowing heterologous production of psilocybin, even when the enzyme has relatively low sequence homology to genes known in the art. The results also show that the introduction of genes from P. cerevisiae into yeast to produce psilocybin is a better strategy than the introduction of genes from P. cerevisiae, with higher production observed. These results also show that cytochrome b5 expression works with cytochrome P450 reductase to further enhance the catalytic activity of tryptamine 4-hydroxylase.

[0263] Example 30 - High level production of psilocybin by genetically modified Saccharomyces cerevisiae In the previous example, a series of substituted tryptamine derivatives were produced by genetically modified Saccharomyces cerevisiae strains. This was achieved by integrating derivatizing enzymes into a genetically modified yeast strain to efficiently produce large amounts of the precursor tryptamine. For example, integration of OsT5H and FoCPR into the high tryptamine producing cell SC-106 resulted in high levels of serotonin production. In this example, genes from D. cerevisiae and D. cerevisiae were used to construct high-producing psilocybin strains by combining beneficial genetic modifications from the previous example. These strains combine various optimized pathways leading to psilocybin, including phosphoketolase bypass (CkPta, BbXfpk, gpp1Δ) to increase erythrose 4-phosphate; erythrose an optimized chorismate pathway (ARO4(K229L), ARO1, ARO2, RIC1Δ) for improved conversion of 4-phosphate and phosphoenolpyruvate to chorismate; an optimized tryptamine pathway (TRP2(S65R, S76L), BsPrs, TRP4) for conversion of chorismate to tryptophan and subsequent decarboxylation to tryptamine, with deletions of ARO10 and PDC5 to eliminate production of tryptophol from tryptophan; , TRP1, TRP3, CrTdc; for the conversion of tryptamine to psilocybin, with a deletion of ERG4 to increase S-adenosylmethionine availability (substrate for methylation by PsiM), and finally, the introduction of an optimized psilocybin pathway (PsiH, CPR, PsiK, PsiM, CYB5) with a deletion of wild-type phosphatase genes (DIA3Δ, PHO5Δ, PHO3Δ) to prevent the degradation of psilocybin to psilocin. During the strain construction process, it became apparent that several biosynthetic pathway steps were rate limiting, and in these cases, overexpression of a second copy of the corresponding genes significantly improved production (ARO4(K229L), TRP2(S65R, S76L), PsiK, PsiM). A summary of all the genetic modifications made resulting in optimized psilocybin producing strains is shown in Table 36. [Table 37]

[0264] Strains were constructed according to Example 1, cultured according to Example 4, and metabolites quantified according to Example 8. As shown in Table 37, the combination of beneficial genetic modifications led to a significant improvement in psilocybin titer, with more than a two-fold improvement in titer and nearly no psilocin accumulation. [Table 38]

[0265] Conclusion: The results of this experiment show that the combination of beneficial genetic modifications resulted in a significant improvement in psilocybin production. These results surprisingly show that different highly genetically modified metabolic pathways can act synergistically to improve overall end-product production. A combinatorial strategy to improve flux through the primary metabolic pathway (tryptophan pathway), reduce flux through competing pathways (pentose phosphate pathway, phenylalanine / tyrosine pathway, ergosterol pathway), improve auxiliary substrate availability (SAM), and remove degradative pathways (tryptophol, psilocin) resulted in a significant improvement in psilocybin production that could only be achieved through this combinatorial genetic modification approach.

[0266] Example 31 - Production of baeocystin by genetically modified Saccharomyces cerevisiae Wild-type psilocybin synthase (PcPsiM, SEQ ID NO: 123) efficiently catalyzes the repeated methylation of norbaeocystin to baeocystin and then to psilocybin. However, a variant of this enzyme is known in the art that catalyzes only the first methylation of norbaeocystin to baeocystin (PcPsiM(H210A), SEQ ID NO: 129). When integrated into Saccharomyces cerevisiae, this variant can be used to produce baeocystin, but not psilocybin. To demonstrate the applicability of this gene variant, the complete psilocybin biosynthetic pathway from Drosophila japonica was integrated into the high-producing tryptamine strain SC-106, but instead of wild-type PcPsiM, the variant PcPsiM(H210A) was integrated. The strain was constructed according to Example 1, cultured according to Example 4, and the resulting metabolites were quantified according to Example 8. The results shown in Table 38 show how integration of PcPsiM(H210) results in the production of only baeocystin, with no psilocybin detected. The dephosphorylated breakdown product of baeocystin, norpsilocin, was also detected, which is to be expected given the high concentrations of baeocystin observed for psilocybin, which is likely a phosphorylated molecule with low stability. [Table 39]

[0267] Conclusion: These results indicate that yeast can also function as a production host to produce this monomethylated intermediate, increasing the need for added expensive substrates and cofactors, as reported for in vitro methods known in the art.

Claims

1. A method for producing a tryptamine derivative of formula (I): 【Chemical 1】 wherein the tryptamine derivative (I) is not tryptophan, 4-hydroxytryptamine, N-acetyl-4-hydroxytryptamine, norbeocystin, beocystin; silocybin, thyrosine, aeruginasin, halogenated tryptophan, halogenated tryptamine, halogenated N-methylated tryptamine, halogenated N,N-dimethyltryptamine or halogenated N,N,N-trimethyltryptamine; Formula (II): 【Chemical 2】 including providing an indole receptor, wherein R ＩＩ , R ＩＶ , R Ｖ , R ＶＩ or R ＶＩＩ one or more of which are not H, and R ＩＩＩ is H; further, in the presence of one or more enzymes that replace one or more of H, OH and / or COOH in the indole receptor with one or more substituents of a substituent donor, contacting the indole receptor with a substituent donor, optionally, one or more of R II, R IV, R V, R VI, and / or R VII of indole receptor (II) are OH, Cl, Br, F, I, CH 3, NO 2, or CH 3 -O, optionally, R 4 and / or R 5 are OH, method.

2. The substituent is an alkyl group, an acetyl group, a glycosyl group, a phosphate group, an oxygenyl group, a hydroxyl group, or a halogenyl group, and optionally, the glycosyl group portion of the glycosyl group comprises one or more of glucose, galactose, xylose, mannose, galactofuranose, arabinose, rhamnose, apiose, fucose, glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine, xylosamine, mannosamine, arabinosamine, rhamnosamine, apirosamine, fucosamine, glucuronic acid, galacturonic acid, mannuronic acid, arabinic acid, apionic acid, or combinations thereof, according to the method of Claim 1.

3. The substitution is selected from the following, according to the method of Claim 2: a) O-alkylation, N-alkylation, or C-alkylation, optionally by ethylation or methylation, b) N-alkylation, and / or c) O-glycosylation, optionally by β-O-glycosylation.

4. The substituent donor is selected from a) aldehydes, ketones, ethers and / or amines, and optionally, i. the aldehyde is acetaldehyde, oxaloacetaldehyde or secologanin, ii. the ketone is cinnamoyl CoA or pyruvic acid, iii. the ether is a glycoside, and optionally, the glycoside is a nucleotide glycoside, and / or iv. the amine is S-adenosylmethionine (SAM) or S-adenosylethionine (SAE), according to the method of Claim 1.

5. One or more of said enzymes are selected from glycosyltransferases, alkyltransferases, synthases, acetyltransferases, kinases, cinnamoyltransferases, phosphatases, laccases, halogenases, P450 enzymes, flavin monooxygenases, and / or lyases, optionally, a) said glycosyltransferase is an O-glycoside transferase and / or a C-glycoside transferase, optionally an aglycon O-glycosyltransferase, glycoside O-glycosyltransferase, aglycon O-glucosyltransferase, aglycon O-rhamnosyltransferase, aglycon O-xylosyltransferase, aglycon O-arabinofuranosyltransferase, aglycon O-N-acetylgalactosaminyltransferase, aglycon O-N-acetylglucosaminyltransferase, aglycon / glycoside mono-O-glycosyltransferase, aglycon / glycoside di-O-glycosyltransferase, aglycon / glycoside tri-O-glycosyltransferase, aglycon / glycoside tetra-O-glycosyltransferase, or hydroxytryptamine glycosyltransferase, optionally a glycosyltransferase contained in any of SEQ ID NOs: 80, 82, 84, 86, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250 and / or 252, and is selected from glycosyltransferases having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity, b) The alkyltransferase is a methyltransferase, optionally an O-methyltransferase, N-methyltransferase or C-methyltransferase, optionally the O-methyltransferase included in any of SEQ ID NOs: 114, 116, 118 and / or 120, and an O-methyltransferase having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity, optionally the N-methyltransferase included in any of SEQ ID NOs: 122, 124, 126, 128, 130, 132, 134, 136 and / or 138, and an N-methyltransferase having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity, or optionally the C-methyltransferase included in SEQ ID NO: 140, and a C-methyltransferase having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity, and is selected from c) The synthase is strictosidine synthase or 1-acetyl-β-carboline synthase, optionally the strictosidine synthase included in any of SEQ ID NOs: 144, 146, 148 and / or 150, and a strictosidine synthase having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity, or optionally the 1-acetyl-β-carboline synthase included in any of SEQ ID NOs: 152 and / or 154, and a 1-acetyl-β-carboline synthase having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity. d) The acetyltransferase is an aralkylamine N-acetyltransferase, optionally an aralkylamine N-acetyltransferase contained in SEQ ID NO: 142, and has at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity with the aralkylamine N-acetyltransferase, e) The kinase is 4-hydroxytryptamine kinase and / or 7-hydroxytryptamine kinase, optionally the 4-hydroxytryptamine kinase and / or 7-hydroxytryptamine kinase contained in any of SEQ ID NOs: 156, 158, and / or 160, and has at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity with the 4-hydroxytryptamine kinase and / or 7-hydroxytryptamine kinase, f) The cinnamoyltransferase is N-hydroxycinnamoyltransferase, optionally the N-hydroxycinnamoyltransferase contained in SEQ ID NO: 162, and has at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity with the N-hydroxycinnamoyltransferase, g) The phosphatase is shirosibirine phosphatase, optionally the shirosibirine phosphatase contained in SEQ ID NO: 164, and has at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity with the shirosibirine phosphatase, h) The laccase is syringol laccase, and optionally, the syringol laccase included in SEQ ID NO: 166 and at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identical to shirosibin laccase, i) The halogenase is tryptophan 2-halogenase, tryptophan 5-halogenase, tryptophan 6-halogenase or tryptophan 7-halogenase, and optionally, i1) the tryptophan 2-halogenase is at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identical to the tryptophan 2-halogenase included in SEQ ID NO: 168, i2) the tryptophan 5-halogenase is at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identical to the tryptophan 5-halogenase included in SEQ ID NO: 170, i3) the tryptophan 6-halogenase is at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identical to the tryptophan 6-halogenase included in SEQ ID NO: 172, or i4) the tryptophan 7-halogenase is at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identical to the tryptophan 7-halogenase included in SEQ ID NO: 174, j) The P450 enzyme is at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identical to the P450 enzyme included in any of SEQ ID NOs: 88, 90, 92, 94, 96, 100 and / or 178, k) The flavin monooxygenase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity with the flavin monooxygenase included in SEQ ID NO: 98, or l) The method according to claim 1, wherein the lyase has at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity with the lyase included in either SEQ ID NO: 52 or 176. **Claim 6** The method according to claim 5, wherein the tryptamine derivative (I) is tyrosine-β-O-glycoside or serotonin-β-O-glycoside. **Claim 7** The following: a) converting an indole or indole derivative to a tryptophan or tryptophan derivative; and b) converting the tryptophan or tryptophan derivative to a tryptamine or tryptamine derivative Optionally further includes one or more steps selected from the above steps in vitro, and optionally i. The conversion of the indole or indole derivative to the tryptophan or tryptophan derivative involves contacting the indole or indole derivative with a tryptophan synthase enzyme, optionally the tryptophan synthase included in SEQ ID NOs: 60, 62, 64, 66, 68, 180, 182 and / or 256, and a tryptophan synthase having at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity. ii. The conversion of the tryptophan or tryptophan derivative to the tryptamine or tryptamine derivative involves contacting the tryptophan or tryptophan derivative with a tryptophan decarboxylase enzyme, optionally the tryptophan decarboxylase included in SEQ ID NO: 26, 70, 72, 74, 76, and / or 78, and a tryptophan decarboxylase having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity, and / or iii. The conversion of the indole or indole derivative to the tryptophan or tryptophan derivative involves contacting the indole or indole derivative with the tryptophan synthase included in SEQ ID NO: 72 and / or 78 and a tryptophan synthase enzyme having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity in the presence of the tryptophan decarboxylase having at least 70%, for example at least 75%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, for example 100% identity. The method according to claim 1 includes this step. **Claim 8** The method according to claim 7, further comprising one or more of the following additional steps: a) Glycosylation; b) Methylation; c) hydroxylation, optionally, the hydroxylation step is, if necessary, in the presence of cytochrome P450 reductase (CPR) contained in any of SEQ ID NOs: 102, 104, 106, 108, 110 and / or 112, and at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity of said CPR, contacting said indole or indole derivative or tryptophan or tryptophan derivative with a hydroxylase, optionally, the hydroxylase contained in any of SEQ ID NOs: 88, 90, 92, 94, 96, 98, and / or 100, and at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity of hydroxylase, including hydroxylation; d) condensation; e) nitration; f) oxidation; g) lyase deamidation, optionally, the lyase deamidation step is contacting said indole or indole derivative or tryptophan or tryptophan derivative with a lyase, optionally, the lyase contained in any of SEQ ID NOs: 52 or 176, and at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, 100% identity of lyase, including lyase deamidation; or h) dephosphorylation.

9. A microbial host cell genetically modified to carry out the method according to claim 1 and produce said tryptamine derivative, expressing one or more heterologous genes encoding one or more enzymes that transfer one or more substituents to one or more of H, OH and / or COOH of said indole acceptor in the presence of said indole acceptor and one or more substituent donors, optionally, said host cell expresses one or more genes selected from the following: a) A gene encoding said UGT, or its genomic DNA, which is at least 70% identical to a UGT-encoding polynucleotide contained in any of SEQ ID NOs: 79, 81, 83, 85, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and / or 251; b) A gene encoding said O-methyltransferase, or its genomic DNA, which is at least 70% identical to an O-methyltransferase-encoding polynucleotide contained in any of SEQ ID NOs: 113, 115, 117 and / or 119; c) A gene encoding said N-methyltransferase, or its genomic DNA, which is at least 70% identical to an N-methyltransferase-encoding polynucleotide contained in any of SEQ ID NOs: 121, 123, 125, 127, 129, 131, 133, 135, and / or 137; d) A gene encoding said C-methyltransferase, or its genomic DNA, which is at least 70% identical to a C-methyltransferase-encoding polynucleotide contained in SEQ ID NO: 139; e) A gene encoding said strictosidine synthase, or its genomic DNA, which is at least 70% identical to a strictosidine synthase-encoding polynucleotide contained in any of SEQ ID NOs: 143, 145, 147 and / or 149; f) A gene encoding said 1-acetyl-β-carboline synthase, or its genomic DNA, which is at least 70% identical to a 1-acetyl-β-carboline synthase-encoding polynucleotide contained in any of SEQ ID NOs: 151 and / or 153; g) A gene encoding said aralkylamine N-acetyltransferase, or its genomic DNA, which is at least 70% identical to a 1-acetyl-β-carboline synthase-encoding polynucleotide contained in SEQ ID NO: 141; h) A gene encoding 4-hydroxytryptamine kinase and / or 7-hydroxytryptamine kinase that is at least 70% identical to the 4-hydroxytryptamine kinase and / or 7-hydroxytryptamine kinase coding polynucleotide contained in any of SEQ ID NO: 155, 157, and / or 159, or their genomic DNA; i) A gene encoding N-hydroxycinnamoyltransferase that is at least 70% identical to the N-hydroxycinnamoyltransferase coding polynucleotide contained in SEQ ID NO: 161, or their genomic DNA; j) A gene encoding sirohydrochlorin phosphatase that is at least 70% identical to the sirohydrochlorin phosphatase coding polynucleotide contained in SEQ ID NO: 163, or their genomic DNA; k) A gene encoding cytosinelaccase that is at least 70% identical to the cytosinelaccase coding polynucleotide contained in SEQ ID NO: 165, or their genomic DNA; l) A gene encoding tryptophan 2-halogenase that is at least 70% identical to the tryptophan 2-halogenase coding polynucleotide contained in SEQ ID NO: 167, or their genomic DNA; m) A gene encoding tryptophan 5-halogenase that is at least 70% identical to the tryptophan 5-halogenase coding polynucleotide contained in SEQ ID NO: 169, or their genomic DNA; n) A gene encoding tryptophan 6-halogenase that is at least 70% identical to the tryptophan 6-halogenase coding polynucleotide contained in SEQ ID NO: 171, or their genomic DNA; o) A gene encoding tryptophan 7-halogenase that is at least 70% identical to the tryptophan 7-halogenase coding polynucleotide contained in SEQ ID NO: 173, or their genomic DNA; p) A gene encoding a P450 enzyme that is at least 70% identical to the P450 enzyme coding polynucleotide contained in any of SEQ ID NO: 87, 89, 91, 93, 95, 99, and / or 177, or their genomic DNA; q) a gene encoding a P450 reductase (CPR) that is at least 70% identical to the P450 reductase coding polynucleotide contained in any of SEQ ID NOs: 101, 103, 105, 107, 109, and / or 111, or their genomic DNA; r) a gene encoding a flavin monooxygenase that is at least 70% identical to the flavin monooxygenase coding polynucleotide contained in SEQ ID NO: 97, or their genomic DNA; and / or s) a gene encoding a lyase that is at least 70% identical to the lyase coding polynucleotide contained in any of SEQ ID NOs: 51 and / or 175, or their genomic DNA, and / or Optionally, the host cell further comprises an effective (operative) biosynthetic pathway for producing the indole receptor and expresses one or more pathway gene coding polypeptides selected from the following: a) one or more enzymes that convert glucose to fructose-6-phosphate; b) fructose-6-phosphate phosphoketolase that converts fructose-6-phosphate to erythrose-4-phosphate and acetyl phosphate; c) phosphotransacetylase that converts acetyl phosphate to acetyl CoA; d) one or more enzymes that convert fructose-6-phosphate to phosphoenolpyruvate; e) 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase) that converts phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP); f) one or more enzymes that convert 3-deoxy-D-arabinoheptulosonate-7-phosphate to 5-enolpyruvoylshikimate 3-phosphate; g) shikimate kinase that converts shikimate to shikimate-3-phosphate; h) chorismate synthase that converts 5-enolpyruvoylshikimate 3-phosphate to chorismate; i) anthranilate synthase that converts chorismate to anthranilic acid; j) ribose-phosphate pyrophosphokinase that converts ribose-5-phosphate to phospho-α-D-ribosyl-1-pyrophosphate; k) Anthranilate phosphoribosyltransferase that converts anthranilic acid and phospho-α-D-ribosyl-1-pyrophosphate to N-(5-phosphoribosyl)-anthranilic acid; l) N-(5'-Phosphoribosyl)anthranilate isomerase that converts N-(5-phosphoribosyl)-anthranilic acid to 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate; m) Phosphoindole-3-glycerol synthase that converts 1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate to (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate; n) Tryptophan synthase that converts (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate and serine to L-tryptophan; o) Tryptophan decarboxylase that converts L-tryptophan to tryptamine; p) Chorismic acid mutase that converts chorismic acid to prephenic acid; q) Prephenic acid dehydrogenase that converts prephenic acid to phenylpyruvic acid; r) Aromatic aminotransferase that converts phenylpyruvic acid to phenylalanine; s) Phenylalanine ammonium lyase that converts phenylalanine to cinnamic acid; t) Cinnamic acid 4-hydroxylase that converts cinnamic acid to coumaric acid; u) Cytochrome b5 that supports cytochrome P450 reductase in reducing the hydroxylase enzyme; v) Cytochrome P450 reductase that reduces the cytochrome P450 enzyme; w) 4-Coumaroyl-CoA ligase that converts coumaric acid to 4-coumaroyl CoA; x) Tryptophanase that converts tryptophan or its derivative to indole or its derivative; y) Tryptophan synthase that converts indole or its derivative and serine or its derivative to tryptophan or its derivative; z) Tryptophan decarboxylase or non-standard aromatic amino acid decarboxylase that converts tryptophan or its derivative to tryptamine or its derivative; aa) Tryptamine 5-hydroxylase that converts tryptamine to serotonin; bb) Tryptamine 4-hydroxylase that converts tryptamine to 4-hydroxytryptamine; cc) 4-Hydroxytryptamine kinase that converts 4-hydroxytryptamine to norbeocystin; and / or dd) Psilocin synthase that converts norbeocystin to psilocin. **Claim 10** One or more wild-type genes are weakened, disrupted, and / or deleted. Optionally, the weakened, disrupted, and / or deleted genes are a) a phosphatase that converts psilocin to cytosine, or b) the host cell according to claim 9, which is a wild-type gene selected from the following: i. A pyruvate kinase gene contained in either SEQ ID NO: 27 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 27; ii. A phosphofructokinase gene contained in either SEQ ID NO: 29 or SEQ ID NO: 31, or a paralog or ortholog thereof having at least 70% identity with either SEQ ID NO: 29 or 31; iii. A transporter gene contained in either SEQ ID NO: 33 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 33; iv. A DL-glycerol-3-phosphate phosphatase gene contained in either SEQ ID NO: 34 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 34; v. A tryptophan 2,3-dioxygenase gene contained in either SEQ ID NO: 35 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 35; vi. A cystathionine β-synthase gene contained in either SEQ ID NO: 36 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 36; vii. A phenylpyruvate decarboxylase gene contained in either SEQ ID NO: 37 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 37; viii. A pyruvate decarboxylase gene contained in either SEQ ID NO: 38 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 38; ix. A histone variant H2AZ gene contained in either SEQ ID NO: 39 or a paralog or ortholog thereof having at least 70% identity with SEQ ID NO: 39; x. A phosphatase gene contained in either SEQ ID NO: 40 or a paralog or ortholog thereof having at least 70% identity thereto; xi. An inhibitory acidic phosphatase gene contained in any one of SEQ ID NO: 41, 42, or 43, or any one of SEQ ID NO: 41, 42, or 43 and a paralog or ortholog thereof having at least 70% identity thereto; xii. A constitutively expressed acidic phosphatase gene contained in either SEQ ID NO: 44 or a paralog or ortholog thereof having at least 70% identity thereto; xiii. A sterol reductase gene contained in either SEQ ID NO: 183 or a paralog or ortholog thereof having at least 70% identity thereto; and / or xiv. An S-adenosylmethionine decarboxylase gene contained in either SEQ ID NO: 184 or a paralog or ortholog thereof having at least 70% identity thereto.

11. A cell culture solution comprising the host cell and growth medium according to Claim 9.

12. The method according to Claim 1, further comprising the following. a) Culturing the cell culture solution according to Claim 11 under conditions that enable the host cell to produce the tryptamine derivative (I); and b) Recovering and / or isolating the tryptamine derivative (I) as necessary.

13. A fermentation broth containing the tryptamine derivative (I) contained in the cell culture solution according to Claim 11.

14. A composition comprising the fermentation broth according to any one of Claim 13 and / or the tryptamine derivative (I) according to Claim 1, and one or more drugs, additives, and / or excipients.

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