Method for producing tryptophan derivatives

Abstract

The present disclosure relates generally to the biosynthetic production of tryptophan derivatives, particularly tryptamine and tryptamine-derived alkaloids.

Description

[Technical Field]

[0001] Related application data This application claims priority from Australian Patent Application No. 2022903920, entitled "Methods for the production of tryptophan derivatives," filed December 20, 2022, and Australian Patent Application No. 2023902441, entitled "Methods for the production of tryptophan derivatives," filed August 2, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is submitted with a Sequence Listing in electronic form, the entire contents of which are incorporated herein by reference.

[0003] Field The present disclosure relates generally to the biosynthetic production of tryptophan derivatives, particularly tryptamine and tryptamine-derived alkaloids. [Background technology]

[0004] The production of secondary metabolites by various fungi, microorganisms, and plants has been an extremely important source of a variety of therapeutically important drugs. Numerous natural products with therapeutic properties or potential therapeutic uses are synthesized from tryptophan and tryptamine, including psilocybin, psilocin, N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenin), serotonin (5-hydroxytryptamine), melatonin, commenecins, ergotamine, harmala alkaloids, and lysergamide. Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine or [3-(2-trimethylaminoethyl)-1H-indol-4-yl] dihydrogen phosphate) is a tryptophan-derived alkaloid abundant in many fungal species. Upon ingestion, psilocybin is dephosphorylated to yield the psychoactive psilocin.

[0005] Unlike many psychoactive substances, psilocybin and psilocin do not appear to be addictive. Psilocybin has been the subject of significant interest regarding its potential use in the treatment of psychiatric disorders. For example, psilocybin is a promising candidate for the treatment of conditions such as depression (including treatment-resistant depression), anxiety disorders, personality disorders, obsessive-compulsive disorder, and substance dependence. Summary of the Invention

[0006] Therefore, there is a need for efficient large-scale production of psilocybin. The psilocybin content of hallucinogenic mushrooms is too low to extract a viable source of the compound. Psilocybin is often produced by complex chemical synthesis. However, this is expensive, time-consuming, and involves the use of irritating, toxic compounds. In addition, the yield and purity of the synthesized compound may be low.

[0007] Pathways for the production of psilocybin and numerous other alkaloid derivatives of tryptophan and tryptamine have been elucidated, and genes involved in the production of intermediates in the pathway have been identified. This opens up the possibility of in vivo production of compounds such as psilocybin from suitable host cells, particularly genetically engineered microorganisms that replicate the natural biosynthetic pathways found in natural fungal and plant sources of the compounds. There is a need in the art for improved methods for increasing the efficiency of synthesis of tryptophan and tryptamine-derived compounds.

[0008] The present disclosure is based on the inventors' surprising identification that fungal pyridoxal phosphate (PLP)-dependent tryptophan decarboxylases from members of the Eurotiomycetes, a class of tryptophan decarboxylases exemplified by tryptophan decarboxylase CnsB from Penicillium expansum and the homologous tryptophan decarboxylase (AsTDC1) from Aspergillus steynii, when expressed in cells such as Saccharomyces cerevisiae, can significantly increase tryptamine production compared to that achieved in S. cerevisiae expressing the PsiD enzyme. Additionally, it was found that tryptophan decarboxylase from Aspergillus caelatus (AcTDC) could significantly increase tryptamine production compared to that achieved with the PsiD enzyme.

[0009] The inventors have also found that PLP-dependent tryptophan decarboxylases, such as those derived from flowering plants, such as CrTDC, can significantly increase tryptamine production compared to that achieved with the PsiD enzyme.

[0010] Based on the above, the present disclosure provides a cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that either lacks the exogenous polynucleotide or comprises a second polynucleotide encoding a second tryptophan decarboxylase, and the second polynucleotide is not the same as the exogenous polynucleotide.

[0011] The present disclosure also provides a cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding a pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that either lacks the exogenous polynucleotide or comprises a second polynucleotide encoding a second tryptophan decarboxylase, wherein the second polynucleotide is not the same as the exogenous polynucleotide, and the PLP-dependent tryptophan decarboxylase comprises one or more or all of the following:

[0012] a) GX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 A first amino acid sequence of R, wherein: X1, X2, X3, X4, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 is any amino acid residue, X5 and X6 are amino acid residues selected from the group consisting of glycine, alanine, serine, and threonine; X7 is an amino acid residue selected from the group consisting of serine and threonine, and X 17 is an amino acid residue selected from the group consisting of alanine and glycine;

[0013] b) X1X2X3X4X5X6HX7X8X9X 10 KX 11 X 12 X 13 X 14 X 15 X 16 a second amino acid sequence of: X1 is any amino acid residue selected from the group consisting of tyrosine, leucine, phenylalanine, valine, cysteine, and tryptophan; X2, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 and X 15 is any amino acid residue, X3 is any amino acid residue selected from the group consisting of serine and threonine; and X 16 is an amino acid residue selected from the group consisting of glycine, alanine, serine and threonine;

[0014] c) DX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 GX 15 X 16 X 17 X 18 X 19 X 20 X 21 A third amino acid sequence of D, wherein: X1, X2, X3, X4, X5, X6, X8, X9, X 10 , X11 , X 12 , X 13 , X 14 , X 15 , X 17 , X 18 , X 19 and X 21 is any amino acid residue, and X7 is an amino acid residue selected from the group consisting of proline and asparagine; X 16 is an amino acid residue selected from the group consisting of threonine, proline, and valine, and X 20 is 1, 2, 3, 4, 5, or 6 amino acid residues, and is any amino acid residue;

[0015] d)X1X2HX3X4X5X6X7X8X9X 10 a fourth amino acid sequence, wherein: X1 is an amino acid residue selected from the group consisting of tryptophan and phenylalanine; X2, X3, X6, X9 and X 10 is any amino acid residue, X4 is an amino acid residue selected from the group consisting of aspartic acid and glutamic acid; X5 is an amino acid residue selected from the group consisting of glycine, alanine, and serine; X7 is an amino acid residue selected from the group consisting of phenylalanine, tyrosine and tryptophan, and X8 is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; and

[0016] e)X1X2X3X4X5X6X7HKX8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 a fifth amino acid sequence of: X1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, histidine, and asparagine; X2 is an amino acid residue selected from the group consisting of serine and threonine; X3, X4, X5, X7, X 10 , X 11 , X 12 , X 13 and X 14 is any amino acid residue, X6 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, and threonine; X8 is an amino acid residue selected from the group consisting of tryptophan, phenylalanine, tyrosine, leucine, methionine, and cysteine; X9 is an amino acid residue selected from the group consisting of leucine, isoleucine, valine, methionine and phenylalanine; X 15 is an amino acid residue selected from the group consisting of cysteine, alanine and serine, and X 16 is an amino acid residue selected from the group consisting of glycine, serine, alanine and threonine.

[0017] In some embodiments, the cells are 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 R, comprising a first amino acid sequence of: X1, X2, X3, X8, X9, X 12 , X 13 , X 14 , X 15 , and X 16 is any amino acid residue, X4 is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X5 and X6 are amino acid residues selected from the group consisting of glycine, alanine, serine, and threonine; X7 is an amino acid residue selected from the group consisting of serine and threonine; X 10 is an amino acid residue selected from the group consisting of asparagine, isoleucine, and valine; X 11 is an amino acid residue selected from the group consisting of leucine, isoleucine, methionine and valine, and X 17 is an amino acid residue selected from the group consisting of alanine and glycine.

[0018] In some embodiments, the cells are 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 R, comprising a first amino acid sequence of: X1, X2, X3, X8, X 12 , X 13 , X 14 , X 15 , and X 16 is any amino acid residue, X4 is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X5 and X6 are amino acid residues selected from the group consisting of glycine and threonine; X7 is an amino acid residue selected from the group consisting of serine and threonine; X9 is an amino acid residue selected from the group consisting of alanine, glycine, cysteine, and serine; X 10 is an amino acid residue selected from the group consisting of asparagine, isoleucine, and valine; X 11 is an amino acid residue selected from the group consisting of leucine, methionine and valine, and X17 is an amino acid residue selected from the group consisting of alanine and glycine.

[0019] In some embodiments, the cells comprise the sequence X1X2X3X4X5X6HX7X8X9X 10 KX 11 X 12 X 13 X 14 X 15 X 16 and a second amino acid sequence of: X1 is any amino acid residue selected from the group consisting of tyrosine, leucine, phenylalanine, valine, cysteine, and tryptophan; X2, X4, X5, X6, X7, X9, X 10 , X 11 , X 12 , X 13 , X 14 and X 15 is any amino acid residue, X3 is any amino acid residue selected from the group consisting of serine and threonine; X8 is any amino acid residue selected from the group consisting of serine and methionine; and X 16 is an amino acid residue selected from the group consisting of glycine, alanine, serine and threonine.

[0020] In some embodiments, the cells comprise the sequence X1X2X3X4X5X6HX7X8X9X 10 KX 11 X 12 X 13 X 14 X 15 X 16 and a second amino acid sequence of: X1 is any amino acid residue selected from the group consisting of tyrosine, leucine, phenylalanine, valine, cysteine, and tryptophan; X2, X4, X7, X9, X 10 , X 11 , X 12 , X 13 , X14 and X 15 is any amino acid residue, X3 is any amino acid residue selected from the group consisting of serine and threonine; X5 is any amino acid residue selected from the group consisting of glutamine and glutamic acid; X6 is any amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X8 is the amino acid residue serine, and X 16 is the amino acid residue glycine.

[0021] In some embodiments, the cells are DX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 GX 15 X 16 X 17 X 18 X 19 X 20 X 21 D, wherein the sequence is: X1, X2, X3, X5, X6, X9, X 10 , X 11 , X 15 , X 17 and X 18 is any amino acid residue, X4 is an amino acid residue selected from the group consisting of glycine, alanine, serine, aspartic acid, and asparagine; X7 is an amino acid residue selected from the group consisting of proline and asparagine; X8 is an amino acid residue selected from the group consisting of phenylalanine, isoleucine, leucine, threonine, tyrosine, and tryptophan; X 12 is an amino acid residue selected from the group consisting of alanine and glycine; X 13is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X 14 is an amino acid residue selected from the group consisting of cysteine, alanine, serine, threonine, leucine, glycine and valine, and X 16 is an amino acid residue selected from the group consisting of threonine, proline, and valine; X 19 is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X 20 is 1, 2, 3, 4, 5, or 6 amino acid residues, and is any amino acid residue; and X 21 is an amino acid residue selected from the group consisting of isoleucine, leucine, valine and threonine.

[0022] In some embodiments, the cells are DX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 GX 15 X 16 X 17 X 18 X 19 X 20 X 21 D, wherein the sequence is: X1, X2, X3, X5, X6, X9, X 11 , X 15 , X 18 and X 20 is any amino acid residue, X4 is an amino acid residue selected from the group consisting of glycine, alanine, serine, aspartic acid, and asparagine; X7 is the amino acid residue proline; X8 is an amino acid residue selected from the group consisting of phenylalanine, threonine, and tyrosine; X 10is an amino acid residue selected from the group consisting of isoleucine, leucine, valine, alanine and glycine; X 12 is an amino acid residue selected from the group consisting of alanine and glycine; X 13 is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X 14 is an amino acid residue selected from the group consisting of cysteine, serine, threonine, leucine, glycine and valine; X 16 is an amino acid residue selected from the group consisting of threonine, proline, and valine; X 17 is an amino acid residue selected from the group consisting of asparagine, lysine, arginine, aspartic acid, serine, and valine; X 19 is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine, and X 21 is an amino acid residue selected from the group consisting of isoleucine, leucine, valine and threonine.

[0023] In some embodiments, the cells comprise the sequence X1X2HX3X4X5X6X7X8X9X 10 and a fourth amino acid sequence of: X1 is an amino acid residue selected from the group consisting of tryptophan and phenylalanine; X2, X3, X6 and X 10 is any amino acid residue, X4 is an amino acid residue selected from the group consisting of aspartic acid and glutamic acid; X5 is an amino acid residue selected from the group consisting of glycine, alanine, and serine; X7 is an amino acid residue selected from the group consisting of phenylalanine, tyrosine, and tryptophan; X8 is an amino acid residue selected from the group consisting of glycine, alanine, serine and threonine, and X9 is an amino acid residue selected from the group consisting of alanine, glycine, leucine, serine and threonine.

[0024] In some embodiments, the cells comprise the sequence X1X2HX3X4X5X6X7X8X9X 10 and a fourth amino acid sequence of: X1 is an amino acid residue selected from the group consisting of tryptophan and phenylalanine; X2 and X3 are any amino acid residues, X4 is the amino acid residue aspartic acid, X5 is an amino acid residue selected from the group consisting of glycine, alanine, and serine; X6 is an amino acid residue selected from the group consisting of alanine, valine, and serine; X7 is an amino acid residue selected from the group consisting of phenylalanine, tyrosine, and tryptophan; X8 is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X9 is an amino acid residue selected from the group consisting of alanine, glycine, leucine, serine and threonine, and X 10 is an amino acid residue selected from the group consisting of serine, alanine, phenylalanine and threonine.

[0025] In some embodiments, the cells comprise the sequence X1X2X3X4X5X6X7HKX8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 and a fifth amino acid sequence of: X1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, histidine, and asparagine; X2 is an amino acid residue selected from the group consisting of serine and threonine; X3, X4, X5, X7, X10 , X 12 and X 14 is any amino acid residue, X6 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, and threonine; X8 is an amino acid residue selected from the group consisting of tryptophan, phenylalanine, tyrosine, and cysteine; X9 is an amino acid residue selected from the group consisting of leucine, isoleucine, valine, methionine and phenylalanine; X 11 is an amino acid residue selected from the group consisting of glutamine, valine, threonine and alanine; X 13 is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, and valine; X 15 is an amino acid residue selected from the group consisting of cysteine, alanine and serine, and X 16 is an amino acid residue selected from the group consisting of glycine, serine, alanine and threonine.

[0026] In some embodiments, the cells comprise the sequence X1X2X3X4X5X6X7HKX8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 and a fifth amino acid sequence of: X1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, histidine, and asparagine; X2 is an amino acid residue selected from the group consisting of serine and threonine; X3, X4 and X 14 is any amino acid residue, X5 is the amino acid residue tryptophan; X6 is the amino acid residue aspartic acid, X7 is an amino acid residue selected from the group consisting of alanine and glycine; X8 is the amino acid residue tryptophan; X9 is the amino acid residue leucine, X 10 is an amino acid residue selected from the group consisting of phenylalanine, leucine, glycine, asparagine, and methionine; X 11 is the amino acid residue glutamine, X 12 is an amino acid residue selected from the group consisting of threonine, serine, leucine, isoleucine, valine, and alanine; X 13 is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, and valine; X 15 is an amino acid residue selected from the group consisting of cysteine ​​and serine, and X 16 is an amino acid residue selected from the group consisting of glycine and serine.

[0027] In some embodiments, the exogenous polynucleotide comprises, in 5' to 3' order, a first amino acid sequence, a second amino acid sequence, a third amino acid sequence, a fourth amino acid sequence, and a fifth amino acid sequence.

[0028] In some embodiments, there are about 2 to about 50 amino acids between the first and second amino acid sequences. In some embodiments, there are about 10 to about 35 amino acids between the first and second amino acid sequences. In some embodiments, there are about 12 to about 25 amino acids between the first and second amino acid sequences.

[0029] In some embodiments, there are about 2 to about 50 amino acids between the second amino acid sequence and the third amino acid sequence. In some embodiments, there are about 20 to about 40 amino acids between the second amino acid sequence and the third amino acid sequence. In some embodiments, there are about 25 to about 35 amino acids between the second amino acid sequence and the third amino acid sequence.

[0030] In some embodiments, there are about 5 to about 50 amino acids between the third amino acid sequence and the fourth amino acid sequence. In some embodiments, there are about 10 to about 25 amino acids between the third amino acid sequence and the fourth amino acid sequence. In some embodiments, there are about 12 to about 22 amino acids between the third amino acid sequence and the fourth amino acid sequence.

[0031] In some embodiments, there are about 5 to about 50 amino acids between the fourth and fifth amino acid sequences. In some embodiments, there are about 10 to about 30 amino acids between the fourth and fifth amino acid sequences. In some embodiments, there are about 15 to about 20 amino acids between the fourth and fifth amino acid sequences.

[0032] The present disclosure also provides a cell comprising an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a fungal pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase from the class Eurotium, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to or contains one or more conservative amino acid substitutions therein, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not harbor the exogenous gene and contains a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical to or contains one or more conservative amino acid substitutions therein.

[0033] The present disclosure also provides a cell comprising an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a PLP-dependent tryptophan decarboxylase (CnsB) having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a sequence having at least about 70% sequence identity thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not harbor the exogenous gene and comprises a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0034] In embodiments, the tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 1, or a sequence that is at least or about 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0035] In embodiments, the cell comprises more than one copy of the exogenous gene, for example, the cell comprises two copies of the exogenous gene.

[0036] The exogenous gene can comprise the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or a sequence having at least about 75% sequence identity thereto. Optionally, the exogenous gene is operably linked to a heterologous promoter. In an exemplary embodiment, the cell comprises two copies of the exogenous gene.

[0037] The present disclosure also provides a cell comprising an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least about 70% sequence identity thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not harbor the exogenous gene and comprises a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0038] The present disclosure also provides a cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that lacks the exogenous polynucleotide or that includes a second polynucleotide encoding a second tryptophan decarboxylase, and the second polynucleotide is not the same as the exogenous polynucleotide.

[0039] In embodiments, the exogenous polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 1, or a sequence that is at least or about 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0040] In embodiments, the exogenous polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 3, or a sequence that is at least or about 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0041] In embodiments, the exogenous polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 5, or a sequence that is at least or about 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0042] In embodiments, the exogenous polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 7, or a sequence that is at least or about 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0043] In embodiments, the exogenous polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 29, or a sequence that is at least or about 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0044] In embodiments, the exogenous polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 48, or a sequence that is at least or about 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0045] In one embodiment, the encoded polypeptide is generated using ancestral sequence reconstruction (e.g., substantially as described in Scossa and Fernie, Computational and Structural Biotechnology Journal, 19:1579-1594, 2021) or by sequence optimization (e.g., substantially as described in Fox and Huisman, Trends in Biotechnology, 26:132-138, 2008 or Chakrababarti et al., Biophysics and Computational Biology, 102:12035-12040, 2005).

[0046] In one embodiment, the encoded polypeptide is generated using directed evolution (eg, substantially as described in Cobb et al., AIChE J. 2013 May;59(5):1432-1440).

[0047] In some embodiments, the exogenous polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 23, 30, 31, 47, and 49-51, or a sequence having at least about 75% sequence identity thereto.

[0048] In some embodiments, the exogenous polynucleotide is operably linked to a heterologous promoter.

[0049] In embodiments, the cell comprises two or more copies of said exogenous polynucleotide, for example, the cell comprises two copies, or three copies, or four copies, or five copies, or six copies, or seven copies of said exogenous polynucleotide.

[0050] In some embodiments, the exogenous polynucleotide is derived from the Eurotium fungi class. In other embodiments, the exogenous polynucleotide is derived from the Dicotyledons class. In other embodiments, the exogenous polynucleotide is derived from the Ascomycota phylum. In other embodiments, the exogenous polynucleotide is derived from the Basidiomycota phylum.

[0051] Those skilled in the art will understand that a polynucleotide as used in this disclosure need not contain the same sequence as that which occurs in nature or from which it is originally derived. For example, a sequence can be codon-optimized for enhanced expression in a cell or organism in which the encoded polypeptide is to be expressed.

[0052] The exogenous gene can comprise the nucleic acid sequence of SEQ ID NO: 8, or a sequence having at least about 75% sequence identity thereto. Optionally, the exogenous gene is operably linked to a heterologous promoter. In an exemplary embodiment, the cell comprises two copies of the exogenous gene.

[0053] In some embodiments, the production level of at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not harbor the exogenous gene and that contains a non-PLP-dependent tryptophan decarboxylase. Optionally, the non-PLP-dependent tryptophan decarboxylase is PsiD. In embodiments, the PsiD tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 9, and optionally, the at least tryptophan derivative is tryptamine or psilocybin.

[0054] In embodiments, the cell can further comprise a nucleic acid sequence encoding an additional tryptophan decarboxylase. The additional tryptophan decarboxylase can be PsiD comprising the amino acid sequence set forth in SEQ ID NO: 9, or a sequence having at least about 70% sequence identity thereto. The additional tryptophan decarboxylase can be CrTDC comprising the amino acid sequence set forth in SEQ ID NO: 29, or a sequence having at least about 70% sequence identity thereto.

[0055] In embodiments, the equivalent cell comprises a polynucleotide encoding a second tryptophan decarboxylase. In some embodiments, the second tryptophan decarboxylase is a PLP-independent tryptophan decarboxylase. For example, the PLP-independent tryptophan decarboxylase is a PsiD tryptophan decarboxylase.

[0056] In embodiments, the cell further comprises at least one polynucleotide encoding an additional tryptophan decarboxylase, for example, the cell further comprises at least two, three, four, five, six, or seven polynucleotides encoding additional tryptophan decarboxylases.

[0057] In some embodiments, the additional tryptophan decarboxylase is CnsBv1. In embodiments, the CnsBv1 tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least about 70% sequence identity thereto.

[0058] In some embodiments, the additional tryptophan decarboxylase is CnsBv2. In embodiments, the CnsBv2 tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least about 70% sequence identity thereto.

[0059] In some embodiments, the additional tryptophan decarboxylase is ΔCnsBv2. In embodiments, the ΔCnsBv2 tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence having at least about 70% sequence identity thereto.

[0060] In some embodiments, the additional tryptophan decarboxylase is asTDC1. In embodiments, the asTDC1 tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least about 70% sequence identity thereto.

[0061] In some embodiments, the additional tryptophan decarboxylase is PsiD. In embodiments, the PsiD tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 9, or a sequence having at least about 70% sequence identity thereto.

[0062] In other embodiments, the additional tryptophan decarboxylase is CrTDC. In embodiments, the CrTDC tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 29, or a sequence having at least about 70% sequence identity thereto.

[0063] In some embodiments, the additional tryptophan decarboxylase is AcTDC. In embodiments, the AcTDC tryptophan decarboxylase comprises the amino acid sequence set forth in SEQ ID NO: 48, or a sequence having at least about 70% sequence identity thereto.

[0064] In an embodiment, the cell can further comprise a nucleic acid sequence encoding a tryptamine 4-monooxygenase comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least about 70% sequence identity thereto. The tryptamine 4-monooxygenase can be PsiH. In an exemplary embodiment, the cell comprises two or more copies of a gene encoding PsiH.

[0065] In embodiments, the cell can further comprise a polynucleotide encoding tryptamine 4-monooxygenase. In such embodiments, the tryptamine 4-monooxygenase is PsiH. In some embodiments, PsiH comprises the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least about 70% sequence identity thereto. In embodiments, the cell comprises two or more copies of a polynucleotide encoding PsiH. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding PsiH.

[0066] In an embodiment, the cell can further comprise a nucleic acid sequence encoding a 4-hydroxytryptamine kinase comprising the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least about 70% sequence identity thereto. The 4-hydroxytryptamine kinase can be PsiK. In an exemplary embodiment, the cell comprises two or more copies of a gene encoding PsiK.

[0067] In embodiments, the cell can further comprise a polynucleotide encoding 4-hydroxytryptamine kinase. In such embodiments, the 4-hydroxytryptamine kinase is PsiK. In some embodiments, PsiK comprises the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least about 70% sequence identity thereto. In embodiments, the cell comprises two or more copies of a polynucleotide encoding PsiK. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding PsiK.

[0068] In embodiments, the cell can further comprise a nucleic acid sequence encoding a methyltransferase comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least about 70% sequence identity thereto. The methyltransferase can be PsiM. In exemplary embodiments, the cell comprises two or more copies of a gene encoding PsiM.

[0069] In embodiments, the cell can further comprise a polynucleotide encoding a methyltransferase. In such embodiments, the methyltransferase is PsiM. In some embodiments, PsiM comprises the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least about 70% sequence identity thereto. In embodiments, the cell comprises two or more copies of a polynucleotide encoding PsiM. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding PsiM.

[0070] In other embodiments, the methyltransferase is RmNMT. In some embodiments, the RmNMT comprises the amino acid sequence set forth in SEQ ID NO: 42, or a sequence having at least about 70% sequence identity thereto. In embodiments, the cell comprises two or more copies of a polynucleotide encoding an RmNMT. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding an RmNMT.

[0071] In an embodiment, the cell can further comprise a nucleic acid sequence encoding a cytochrome P450 reductase comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto. The cytochrome P450 reductase can be PcCpr. In an exemplary embodiment, the cell comprises two or more copies of a gene encoding PcCpr.

[0072] In embodiments, the cell may further comprise a polynucleotide encoding a cytochrome P450 reductase. In such embodiments, the cytochrome P450 reductase is PcCpr. In some embodiments, PcCpr comprises the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto. In embodiments, the cell comprises two or more copies of a polynucleotide encoding PcCpr. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding PcCpr.

[0073] In other embodiments, the cytochrome P450 reductase is OsCPR. In some embodiments, OsCPR comprises the amino acid sequence set forth in SEQ ID NO: 39, or a sequence having at least about 70% sequence identity thereto. In embodiments, the cell comprises two or more copies of a polynucleotide encoding OsCPR. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding OsCPR.

[0074] In embodiments, the cells may further comprise one or more, optionally at least two copies of a gene encoding an adenosylhomocysteinase. The adenosylhomocysteinase may be Sah1. In embodiments, the cells may further comprise one or more, optionally at least two copies of a gene encoding an adenosine kinase. The adenosine kinase may be Ado1.

[0075] In embodiments, the cell can further comprise at least one copy of a polynucleotide encoding an adenosylhomocysteinase. In some embodiments, the adenosylhomocysteinase is Sah1. In some embodiments, Sah1 comprises the amino acid sequence set forth in SEQ ID NO: 19, or a sequence having at least about 70% sequence identity thereto. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding Sah1.

[0076] In embodiments, the cell can further comprise at least one copy of a polynucleotide encoding an adenosine kinase. In some embodiments, the adenosine kinase is Ado1. In some embodiments, Ado1 comprises the amino acid sequence set forth in SEQ ID NO: 21, or a sequence having at least about 70% sequence identity thereto. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding Ado1.

[0077] In embodiments, the cell may further comprise at least one copy of a polynucleotide encoding tryptamine 5-hydroxylase. In some embodiments, the tryptamine 5-hydroxylase is OsT5H. In some embodiments, the OsT5H comprises the amino acid sequence set forth in SEQ ID NO: 36, or a sequence having at least about 70% sequence identity thereto. In some embodiments, the cell comprises at least two, three, four, five, six, or seven polynucleotides encoding OsT5H.

[0078] In embodiments, the cell is a yeast cell. In embodiments, the yeast cell is a Saccharomyces sp. cell, e.g., a S. cerevisiae cell. In embodiments, the yeast cell is a Yarrowia sp. cell.

[0079] In an embodiment, the cell is a bacterial cell. In an embodiment, the bacterial cell is an Escherichia sp., such as E. coli. In an embodiment, the bacterial cell is a Corynebacterium sp., such as C. glutamicum.

[0080] In an embodiment, the cell is a fungal cell. In an embodiment, the fungal cell is an Aspergillus sp.

[0081] In embodiments, the at least one tryptophan derivative may be tryptamine or a tryptamine-derived alkaloid. In an exemplary embodiment, the tryptophan derivative is tryptamine. In another embodiment, the tryptamine-derived alkaloid is psilocybin. In another embodiment, the tryptophan derivative is serotonin. In another embodiment, the tryptophan derivative is bufotenin (5-hydroxy-N,N-dimethyltryptamine). In another embodiment, the tryptophan derivative is N,N-dimethyl-tryptamine. In another embodiment, the tryptophan derivative is 5-methoxy-N,N-dimethyltryptamine. In another embodiment, the tryptophan derivative is N-methyltryptamine. In another embodiment, the tryptophan derivative is N-acetylserotonin. In another embodiment, the tryptophan derivative is melatonin.

[0082] The present disclosure also provides (a) one or more copies of a gene encoding a fungal PLP-dependent tryptophan decarboxylase from the class Eurotium, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to, or contains one or more conservative amino acid substitutions relative to, said fungal PLP-dependent tryptophan decarboxylase; (b) one or more copies of a gene encoding PsiH tryptamine 4-monooxygenase; (c) one or more copies of a gene encoding the PsiK hydroxytryptamine kinase; and (d) one or more copies of a gene encoding a PsiM methyltransferase Also provided is a cell capable of producing at least one tryptophan derivative, comprising:

[0083] The present disclosure also provides (a) one or more copies of a gene encoding a PLP-dependent tryptophan decarboxylase (CnsB) having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a sequence having at least about 70% sequence identity thereto; (b) one or more copies of a gene encoding PsiH tryptamine 4-monooxygenase; (c) one or more copies of a gene encoding the PsiK hydroxytryptamine kinase; and (d) one or more copies of a gene encoding a PsiM methyltransferase Also provided is a cell capable of producing at least one tryptophan derivative, comprising:

[0084] The present disclosure also provides (a) one or more copies of a gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least about 70% sequence identity thereto; (b) one or more copies of a gene encoding PsiH tryptamine 4-monooxygenase; (c) one or more copies of a gene encoding the PsiK hydroxytryptamine kinase; and (d) one or more copies of a gene encoding a PsiM methyltransferase Also provided is a cell capable of producing at least one tryptophan derivative, comprising:

[0085] The present disclosure also provides (a) at least one exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase as described herein; (b) at least one exogenous polynucleotide encoding a PsiH tryptamine 4-monooxygenase; (c) at least one exogenous polynucleotide encoding a PsiK hydroxytryptamine kinase; and (d) at least one exogenous polynucleotide encoding a PsiM methyltransferase; Also provided is a cell capable of producing at least one tryptophan derivative, comprising:

[0086] The present disclosure also provides (a) at least one exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto; (b) at least one exogenous polynucleotide encoding a PsiH tryptamine 4-monooxygenase; (c) at least one exogenous polynucleotide encoding a PsiK hydroxytryptamine kinase; and (d) one or two copies of an exogenous polynucleotide encoding a PsiM methyltransferase Also provided is a cell capable of producing at least one tryptophan derivative, comprising:

[0087] The present disclosure also provides (a) at least one exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase described herein; (b) one or more copies of an exogenous polynucleotide encoding OsT5H tryptamine 5-hydroxylase; (c) one or more copies of an exogenous polynucleotide encoding an OsCPR cytochrome P450 reductase; and (d) one or more copies of an exogenous polynucleotide encoding an RmNMT methyltransferase Also provided is a cell capable of producing at least one tryptophan derivative, comprising:

[0088] The present disclosure also provides (a) at least one exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO: 7; (b) at least one exogenous polynucleotide encoding an OsT5H tryptamine 5-hydroxylase; (c) at least one exogenous polynucleotide encoding an OsCPR cytochrome P450 reductase; and (d) at least one exogenous polynucleotide encoding an RmNMT methyltransferase; Also provided is a cell capable of producing at least one tryptophan derivative, comprising:

[0089] In some embodiments, the cells are capable of a higher level of production of at least one tryptophan derivative compared to the level of production of the at least one tryptophan derivative in a comparable cell that does not possess (a) and includes a non-PLP-dependent tryptophan decarboxylase, optionally the non-PLP-dependent tryptophan decarboxylase is PsiD. Optionally, the tryptophan derivative is tryptamine or psilocybin. Optionally, the tryptophan derivative is serotonin, bufotenin (5-hydroxy-N,N-dimethyltryptamine), N,N-dimethyl-tryptamine, 5-methoxy-N,N-dimethyltryptamine, N-methyltryptamine, N-acetylserotonin, or melatonin.

[0090] In some embodiments, the cell is capable of an increased level of production of at least one tryptophan derivative compared to the level of production of the second tryptophan derivative in an equivalent cell comprising a second polynucleotide encoding a second tryptophan decarboxylase, wherein the second polynucleotide is not the same as (a). In some embodiments, the second polynucleotide encodes a PsiD tryptophan decarboxylase.

[0091] The cells may further comprise one or more copies of a gene encoding a cytochrome P450 reductase, optionally a PcCpr cytochrome P450 reductase. The cells may further comprise one or more copies of a gene encoding an adenosylhomocysteinase, optionally a Sah1 adenosylhomocysteinase. The cells may further comprise one or more copies of a gene encoding an adenosine kinase, optionally an Ado1 adenosine kinase.

[0092] The cells may further comprise one or more copies of a gene encoding an additional tryptophan decarboxylase, optionally PsiD and / or CrTDC tryptophan decarboxylase.

[0093] One skilled in the art will understand that all embodiments of the cells described herein relate to any of the cells described herein.

[0094] The present disclosure also provides a nucleic acid construct or vector comprising a nucleic acid sequence encoding a fungal PLP-dependent tryptophan decarboxylase from the class Eurotium, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to or comprises one or more conservative amino acid substitutions thereto, optionally wherein the nucleic acid sequence is operably linked to a heterologous promoter, and the tryptophan decarboxylase encoded by the gene is capable of producing a higher level of at least one tryptophan derivative in a cell compared to the production level of at least one tryptophan derivative in an equivalent cell that does not possess the gene and comprises a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical to or comprises one or more conservative amino acid substitutions thereto.

[0095] The present disclosure also provides a nucleic acid construct or vector comprising the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:23, or a sequence having at least about 75% sequence identity thereto, optionally operably linked to a heterologous promoter, wherein the tryptophan decarboxylase encoded by said gene is capable of a higher level of production of at least one tryptophan derivative in a cell compared to the level of production of at least one tryptophan derivative in an equivalent cell that does not possess said gene and comprises a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0096] The present disclosure also provides a nucleic acid construct or vector comprising the nucleic acid sequence of SEQ ID NO: 8, or a sequence having at least about 75% sequence identity thereto, optionally operably linked to a heterologous promoter, wherein the tryptophan decarboxylase encoded by the gene is capable of producing a higher level of at least one tryptophan derivative in a cell compared to the production level of at least one tryptophan derivative in an equivalent cell that does not possess the gene and comprises a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0097] The present disclosure also provides vectors comprising a polynucleotide encoding a PLP-dependent tryptophan decarboxylase as described herein, optionally operably linked to a heterologous promoter capable of directing expression of the polynucleotide. In some embodiments, the polynucleotide is capable of increased production levels of at least one tryptophan derivative in a cell compared to the production level of the at least one tryptophan derivative in an equivalent cell lacking the polynucleotide or comprising a second polynucleotide encoding a second tryptophan decarboxylase, and the second polynucleotide is not the same as the polynucleotide.

[0098] The present disclosure also provides a vector comprising a polynucleotide encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, and 7, optionally operably linked to a heterologous promoter capable of directing expression of the polynucleotide in a cell that does not naturally express the polynucleotide. In some embodiments, the polynucleotide is capable of increased production levels of at least one tryptophan derivative in a cell compared to the production level of the at least one tryptophan derivative in an equivalent cell lacking the polynucleotide or comprising a second polynucleotide encoding a second tryptophan decarboxylase, and the second polynucleotide is not identical to the polynucleotide.

[0099] The present disclosure also provides a vector comprising a polynucleotide encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 29 and 48, optionally wherein the polynucleotide is operably linked to a heterologous promoter capable of directing expression of the polynucleotide in a cell that does not naturally express the polynucleotide. In some embodiments, the polynucleotide is capable of increased production levels of at least one tryptophan derivative in a cell compared to the production level of the at least one tryptophan derivative in an equivalent cell lacking the polynucleotide or comprising a second polynucleotide encoding a second tryptophan decarboxylase, and the second polynucleotide is not the same as the polynucleotide.

[0100] In embodiments, the non-PLP-dependent tryptophan decarboxylase is PsiD, optionally comprising the amino acid sequence set forth in SEQ ID NO: 9. Optionally, the at least tryptophan derivative is tryptamine or psilocybin.

[0101] The present disclosure also provides a host cell comprising a nucleic acid construct or vector of the present disclosure.

[0102] The present disclosure also provides a method for producing at least one tryptophan derivative, comprising culturing a cell of the present disclosure under conditions suitable for the production of at least one tryptophan derivative.

[0103] The method can further include extracting at least one tryptophan derivative from the cells and / or the medium in which the cells are cultured. The at least one tryptophan derivative can be tryptamine or a tryptamine-derived alkaloid. In an exemplary embodiment, the tryptophan derivative is tryptamine. In another embodiment, the tryptamine-derived alkaloid is psilocybin.

[0104] The present disclosure also provides a method for producing at least one tryptophan derivative, comprising contacting tryptophan with at least one pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase, as described herein, under conditions suitable for the production of at least one tryptophan derivative.

[0105] The present disclosure also provides a method for producing at least one tryptophan derivative, comprising contacting tryptophan with at least one pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto, under conditions suitable for the production of the at least one tryptophan derivative.

[0106] In one example, the method is a cell-free method or is performed using a cell lysate, for example, from a cell as described herein.

[0107] In some embodiments, the method is carried out in a bioreactor.

[0108] In some embodiments, the polypeptide and / or tryptophan is provided in a cell lysate. In some embodiments, the polypeptide is provided as a purified and / or isolated polypeptide.

[0109] The present disclosure also provides tryptophan derivatives produced by the methods described herein.

[0110] The present disclosure also provides compositions comprising the cells of the present disclosure or tryptophan derivatives extracted therefrom.

[0111] The present disclosure also provides an isolated nucleotide sequence comprising a gene encoding a fungal PLP-dependent tryptophan decarboxylase from the class Eurotium, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to the fungal PLP-dependent tryptophan decarboxylase or contains one or more conservative amino acid substitutions thereto, operably linked to a heterologous promoter capable of directing expression of the gene in a cell that does not naturally express the gene.

[0112] The present disclosure also provides an isolated nucleotide sequence comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a sequence having at least about 70% sequence identity thereto, operably linked to a heterologous promoter capable of directing expression of said gene in a cell that does not naturally express said gene.

[0113] The present disclosure also provides an isolated nucleotide sequence comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto, operably linked to a heterologous promoter capable of directing expression of said gene in a cell that does not naturally express said gene.

[0114] The present disclosure also provides an isolated nucleotide sequence comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NOs: 29 and 48, or a sequence having at least about 70% sequence identity thereto, operably linked to a heterologous promoter capable of directing expression of said gene in a cell that does not naturally express said gene.

[0115] The present disclosure also provides a method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of the present disclosure. Optionally, the disease or disorder is a neurodevelopmental, neurological, neuromuscular, movement, psychiatric, or psychological disease, disorder, or syndrome. In exemplary embodiments, the disease or disorder is selected from depression or a depressive disorder, anxiety disorder, obsessive-compulsive disorder, personality disorder, substance addiction or dependence, post-traumatic stress disorder, migraine, and / or chronic headache.

[0116] The present disclosure also provides compositions of the present disclosure for use or administration as a nootropic or functional food.

[0117] The present disclosure also provides use of a cell of the present disclosure, or a tryptophan derivative extracted therefrom, in the manufacture of a medicament for treating or preventing a disease or disorder in a subject. Optionally, the disease or disorder is a neurodevelopmental, neurological, neuromuscular, movement, psychiatric, or psychological disease, disorder, or syndrome. In some embodiments, the medicament is provided as a nootropic. In other embodiments, the medicament is provided as a functional food.

[0118] Embodiments of the present disclosure are herein described, by way of non-limiting example only, with reference to the following figures: [Brief explanation of the drawings]

[0119] [Figure 1] FIG. 1 shows a biosynthetic pathway for the production of psilocybin and psilocin from L-tryptophan. [Figure 2]Pairwise Needleman-Wunsch alignment of the polypeptide sequences of (A) CnsBv1 (SEQ ID NO: 1) and CnsBv2 (SEQ ID NO: 3); and (B) CnsBv1 (SEQ ID NO: 1) and ΔCnsBv2 (SEQ ID NO: 5). Conserved residues are shown in the line between the two sequences. [Figure 3] Pairwise Needleman-Wunsch alignment of the polypeptide sequences of (A) CnsBv1 (SEQ ID NO: 1) and PsiD (SEQ ID NO: 9); and (B) CnsBv2 (SEQ ID NO: 3) and PsiD (SEQ ID NO: 9). Conserved residues are shown in the line between the two sequences. [Figure 4] Figure 4 shows pairwise Needleman-Wunsch alignments of the polypeptide sequences of (A) CnsBv1 (SEQ ID NO: 1) and CrTDC (SEQ ID NO: 29); and (B) CnsBv2 (SEQ ID NO: 3) and CrTDC (SEQ ID NO: 29). Conserved residues are shown in the line between the two identified sequences. [Figure 5] Figure 5 shows pairwise Needleman-Wunsch alignments of the polypeptide sequences of (A) AsTDC1 (SEQ ID NO: 7) and CnsBv1 (SEQ ID NO: 1); (B) AsTDC1 (SEQ ID NO: 7) and CnsBv2 (SEQ ID NO: 3); (C) AsTDC1 (SEQ ID NO: 7) and PsiD (SEQ ID NO: 9); and (D) AsTDC1 (SEQ ID NO: 7) and CrTDC (SEQ ID NO: 29). Conserved residues are shown in the line between the two identified sequences. [Figure 6] FIG. 6 shows tryptamine yields (mg / L) from cells (A) and growth medium (B) of S. cerevisiae strains SC-cnsBv1 and SC-psiD measured after 5 and 6 days of culture. [Figure 7] FIG. 7 shows tryptamine yields (mg / L) from S. cerevisiae cells SC-cnsBv1, SC-asTDC1, and SC-psiD measured after 3, 5, and 6 days of culture. [Figure 8]FIG. 8 shows the combined psilocybin and psilocin yields (mg / L) from cells (A) and growth medium (B) of S. cerevisiae strains SC-D and SC-B measured after 2 and 3 days of culture. [Figure 9] FIG. 9 shows the combined psilocybin and psilocin yields (mg / L) from cells (A) and growth medium (B) of S. cerevisiae strains SC-DM and SC-BM measured after 2 and 3 days of culture. [Figure 10] FIG. 10 shows the combined psilocybin and psilocin yields (mg / L) from cells (A) and growth medium (B) of S. cerevisiae strains SC-DMAS and SC-BMAS measured after 2 and 3 days of culture. [Figure 11] FIG. 11 shows the combined psilocybin and psilocin yields (mg / L) from cells (A) and growth medium (B) of S. cerevisiae strains SC-DMASB and SC-BMASB measured after 2 and 3 days of culture. [Figure 12] FIG. 12 shows the combined psilocybin and psilocin yields (mg / L) from cells and growth medium of S. cerevisiae strains SC-D, SC-B, SC-DM, SC-BM, SC-DMAS, SC-BMAS, SC-DMASB, and SC-BMASB measured after 3 days of culture. [Figure 13] FIG. 13 shows the biosynthetic pathway from tryptophan to N,N-dimethyltryptamine and bufotenin. [Figure 14] Figure 14 shows the relative compound abundances of metabolite extractions for S. cerevisiae strains (A) SC-rmNMT1, (B) SC-rmNMT2, (C) SC-rmNMT3, (D) SC-rmNMT4, and (E) SC-rmNMT5, measured after 6 days of cultivation. Average peak areas of extracted ion chromatograms from three biological replicates per strain are shown for tryptamine, 5-OH-tryptophan, serotonin, N,N-dimethyltryptamine, and bufotenin. Individual gene combinations for each strain are indicated below their respective bar graphs. [Figure 15]FIG. 15 shows the tryptamine yields of E. coli strains EC-psiD, EC-asTDC1 and EC-ctrl measured after 3 days of cultivation. [Figure 16] FIG. 16 shows the tryptamine yield of six strains of S. cerevisiae expressing different TDCs on day 3 of culture. DETAILED DESCRIPTION OF THE INVENTION

[0120] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications, published applications and publications, databases, websites, and other published materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise noted. In the event of multiple definitions of a term, those in this section prevail. When reference is made to a URL or other such identifier or address, it is understood that such identifiers may change and particular information on the Internet may come and go, but that equivalent information can be found by searching the Internet. Reference to an identifier evidences the availability and public dissemination of such information.

[0121] As used herein, the singular forms "a," "an," and "the" include plural references (i.e., at least one or more than one) unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a single polypeptide as well as two or more polypeptides.

[0122] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative (or).

[0123] In the context of this specification, the term "about" is understood to mean a range of numbers that one of ordinary skill in the art would consider equivalent to the specified value, in the context that achieves the same function or result.

[0124] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements cannot be present.

[0125] As used herein, the term "exogenous" in the context of a gene or polynucleotide refers to the gene or polynucleotide when present in a cell that does not naturally contain the gene or polynucleotide. An exogenous gene or polynucleotide can include a promoter and / or other regulatory genetic elements from its original source, or can be operably linked to one or more heterologous regulatory elements. Such heterologous regulatory elements can be endogenous to the cell (cell) or can themselves be exogenous.

[0126] The term "operably linked," as used herein, refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, this term refers to the functional relationship of a transcriptional regulatory element (promoter) to a transcribed sequence, such as an exogenous gene as described herein. For example, a promoter is operably linked to a gene coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located adjacent to the coding sequence whose transcription they enhance. For example, introns in 5'UTR sequences or toward the 5' end of a protein coding region can contain transcriptional enhancers to result in increased expression levels, e.g., the FBAIN promoter region.

[0127] As used herein, the term "pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase" means a tryptophan decarboxylase that requires the active site binding of pyridoxal 5' phosphate (PLP) for catalytic activity, i.e., for catalyzing the conversion of tryptophan to tryptamine.

[0128] As used herein, the term "derived" in the context of a fungal pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase derived from the class Eurotiomycete includes PLP-dependent tryptophan decarboxylases obtained from fungal organisms belonging to the class Eurotiomycete, and variants of such PLP-dependent tryptophan decarboxylases. The PLP-dependent tryptophan decarboxylase may be the wild-type or native enzyme found in the class Eurotiomycete, or a mutant, derivative, or variant thereof. The terms "wild-type," "native," and "naturally occurring" are used interchangeably herein to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type, native, or naturally occurring gene or gene product (e.g., a protein or polypeptide) is one that is most frequently observed in a population and is thus the arbitrarily designed "normal" or "wild-type" form of the gene or gene product. As will be understood by those skilled in the art, the terms "fungal PLP-dependent tryptophan decarboxylase," "fungal PLP-dependent tryptophan decarboxylase from the class Eurotium" and "PLP-dependent tryptophan decarboxylase from the class Eurotium" are used interchangeably herein.

[0129] As will be understood by one of skill in the art, the terms "pyridoxal phosphate (PLP)-independent tryptophan decarboxylase" and "non-PLP-dependent tryptophan decarboxylase" can be used interchangeably and refer to a tryptophan decarboxylase that does not require active site binding of pyridoxal 5' phosphate (PLP) for catalytic activity.

[0130] As used herein, the terms "variant" and "variants" refer to substantially similar sequences. Variants can be naturally occurring or artificially generated. A variant of a fungal PLP-dependent tryptophan decarboxylase can share at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the fungal PLP-dependent tryptophan decarboxylase of which it is a variant, such as a decarboxylase comprising the amino acid sequence of any one of SEQ ID NOs: 1, 3, 5, or 7. Generally, variant polypeptides and proteins also retain common qualitative biological activities, particularly catalytic activity. Variants of the fungal PLP-dependent tryptophan decarboxylase of the present disclosure can contain one or more conservative amino acid substitutions relative to the fungal PLP-dependent tryptophan decarboxylase.

[0131] A variant of a PLP-dependent tryptophan decarboxylase can share at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the PLP-dependent tryptophan decarboxylase of which it is a variant, such as a decarboxylase comprising the amino acid sequence of any one of SEQ ID NOs: 29 or 48. Generally, variant polypeptides and proteins also retain common qualitative biological activities, particularly catalytic activity. A variant of a fungal PLP-dependent tryptophan decarboxylase of the present disclosure can include one or more conservative amino acid substitutions relative to a PLP-dependent tryptophan decarboxylase.

[0132] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that replacement of leucine with isoleucine or valine, replacement of aspartic acid with glutamic acid, replacement of threonine with serine, or similar replacement of an amino acid with a structurally related amino acid will not have a significant effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity.

[0133] Suitable examples of amino acid modifications to tryptophan decarboxylase are set forth in US2023 / 0357744 and WO2021 / 248087. It will be understood that the scope of the present disclosure encompasses such amino acid modifications.

[0134] In some embodiments, the amino acid alterations to the tryptophan decarboxylases described herein are generated by directed evolution (eg, non-natural sequences generated by sequence optimization).

[0135] The terms "protein" and "polypeptide" are used interchangeably herein. A polypeptide or protein having non-polypeptide moieties covalently or non-covalently associated therewith is still considered a "polypeptide." Exemplary modifications include glycosylation and palmitoylation. Polypeptides and proteins can be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid-phase peptide synthesis, etc. The term "polypeptide sequence" or "amino acid sequence," as used herein, can refer to the polypeptide material itself and / or sequence information that biochemically characterizes the polypeptide (e.g., a series of letters or three-letter code used as an abbreviation for the amino acid designation). Polypeptide sequences presented herein are presented in an N-terminal to C-terminal direction unless otherwise indicated.

[0136] The term "polynucleotide" is used interchangeably herein with "nucleic acid," "nucleic acid molecule," and "nucleotide sequence" and "gene" to refer to a polymer of nucleosides. Typically, polynucleotides of the present disclosure are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term also encompasses molecules containing nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for particular applications. When the present application refers to polynucleotides, it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and the complement of each single-stranded molecule) are provided. "Polynucleotide sequence" or "nucleotide sequence," as used herein, can refer to the polynucleotide material itself and / or the sequence information (e.g., a series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise indicated.

[0137] The term "nucleic acid construct" refers to a recombinant genetic molecule containing one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule, and includes any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences, that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), or (2) sequences encoding portions of functional RNA molecules or proteins that are not naturally contiguous, or (3) portions of promoters that are not naturally contiguous. Exemplary constructs include any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single- or double-stranded DNA or RNA nucleic acid molecule from any source, capable of genome integration or autonomous replication, that contains a nucleic acid molecule to which one or more nucleic acid molecules are operably linked. Constructs of the present disclosure will generally include the necessary elements for directing expression of a nucleic acid sequence of interest also included in the construct, such as a target nucleic acid sequence or a regulatory nucleic acid sequence. Such elements may include a control element, such as a promoter, operably linked to the nucleic acid sequence of interest (to direct its transcription) and often including a polyadenylation sequence. Within certain embodiments of the present disclosure, the construct may be included within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements for facilitating stable integration of the construct into the genome of a host cell. Two or more constructs may be included within a single nucleic acid molecule, such as a single vector, or may be included within two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to a nucleotide sequence of interest.In this manner, for example, a promoter in operable linkage with a nucleotide sequence to be expressed is provided in an expression construct for expression in an organism or portion thereof, including a host cell. Conventional compositions and methods for preparing and using constructs and host cells for practicing the methods of the present disclosure are well known to those of skill in the art; see, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, Vols. 1, 2, and 3, J.F. Sambrook, D.W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0138] As used herein, the term "derivative" in the context of tryptophan derivatives means a compound that is biosynthetically produced either directly or indirectly from tryptophan in one or more enzymatically catalyzed steps, i.e., the compound is part of the same biosynthetic pathway as tryptophan and is downstream of tryptophan in that biosynthetic pathway.

[0139] Similarly, the term "derived" in the context of a tryptamine-derived compound or alkaloid as described herein means a compound that is biosynthetically produced either directly or indirectly from tryptamine in one or more enzyme-catalyzed steps, i.e., the compound is part of the same biosynthetic pathway as tryptamine and is downstream from tryptamine in that biosynthetic pathway.

[0140] As used herein, "vector" includes reference to both polynucleotide vectors and viral vectors, each of which is capable of delivering a transgene contained within the vector into a host cell. Vectors can be episomal, i.e., not integrating into the genome of a host cell, or can be integrated into the host cell genome. Vectors can also be replication competent or replication defective. Exemplary polynucleotide vectors include, but are not limited to, plasmids, cosmids, and transposons. Exemplary viral vectors include, for example, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus vectors.

[0141] As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, enhancer, or set of transcription factor binding sites) and a coding sequence (e.g., of an exogenous gene as described herein) wherein the expression control sequence affects the transcription and / or translation of the coding sequence.

[0142] The term "host cell" means a cell, such as a yeast cell, into which an exogenous gene as described herein, such as in a vector or other polynucleotide, has been introduced. The term includes the progeny of the original cell into which the exogenous gene was introduced. Thus, "host cell," as used herein, generally refers to a cell that has been transfected or transduced with exogenous DNA.

[0143] As used herein, "isolated" with respect to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is substantially free of cellular material or other contaminating proteins from the cell from which the polynucleotide or polypeptide is derived, or, if chemically synthesized, is substantially free of chemical precursors or other chemicals. As used herein, the term "purified," when used in connection with the tryptophan derivatives disclosed herein that are extracted from cells or cell growth or culture medium, means that the extracted tryptophan derivative has been subjected to one or more processing steps that increase the purity of the tryptophan derivative.

[0144] As used herein, the term "subject" includes any mammal, such as a human, non-human primate, livestock animal (e.g., sheep, pigs, cows, horses, donkeys, goats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, other rodents), companion animals (e.g., dogs, cats), etc. In a preferred embodiment, the subject is a human.

[0145] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect in a subject in need of treatment, i.e., a subject having a disease or disorder. "Treatment" means ameliorating or preventing one or more symptoms or effects (e.g., prognosis) of a disease or disorder. Reference to "treatment," "treat," or "treating" does not necessarily mean reversing or preventing any or all symptoms or effects of a disease or disorder. For example, a subject may ultimately suffer from one or more symptoms or effects, but the number and / or severity of the symptoms or effects is reduced and / or the quality of life is improved compared to before treatment.

[0146] As used herein, the term "nootropic" will be understood to mean any natural, semi-synthetic, biosynthetic, and synthetic neuroprotective or cognitive enhancing compound.

[0147] As used herein, the term "functional food" will be understood to mean a consumable product that can beneficially alter one or more target functions in a subject (e.g., by enhancing a physiological response and / or reducing the risk of disease).

[0148] It will be understood that the above terms and associated definitions are used for purposes of description only and are not intended to be limiting.

[0149] Each embodiment described herein should be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0150] [Table 1-1] [Table 1-2]

[0151] PLP-dependent tryptophan decarboxylase-expressing cells With the elucidation of the biosynthetic pathways for the production of tryptophan-derived secondary metabolites and the genes involved in the production of various intermediates in such pathways, some attention has been directed to manipulating these pathways in an attempt to increase or improve the production of various tryptophan derivatives, including using heterologous in vivo expression of one or more enzymes operating in the pathway in suitable host cells. To date, most attention has focused on the use of the PsiD, PsiH, PsiK, and PsiM enzymes, particularly those from Psilocybe cubensis (e.g., PcPsiD, PcPsiH, PcPsiK, and PcPsiM), for the production of psilocybin.

[0152] The biosynthetic pathway for the production of psilocybin from tryptophan, illustrating the roles of the PsiD, PsiH, PsiK, and PsiM enzymes, is shown in Figure 1. PsiD is a PLP-independent tryptophan decarboxylase that catalyzes the decarboxylation of aliphatic carboxylic acids, converting L-tryptophan to tryptamine. PsiH is a monooxygenase that catalyzes the oxidative hydroxylation of the phenyl ring of tryptamine to 4-hydroxytryptamine. PsiH belongs to the superfamily of heme-containing monooxygenases, which typically require a cytochrome P450 reductase partner (CPR) for efficient catalysis. PsiK is a kinase that catalyzes the phosphorylation of the phenolic oxygen of 4-hydroxytryptamine to norbeocystin and the subsequent phosphorylation of psilocybin to psilocybin. PsiM is a methyltransferase that catalyzes the alkylation of primary amines in norbaeocystin to baeocystin and secondary amines of baeocystin to tertiary amines of psilocybin. U.S. Patent No. 11,441,164 describes a method for the production of psilocybin in recombinant organisms expressing genes encoding PsiD, PsiH, PsiK, and PsiM that have been codon-optimized for expression in a host organism.

[0153] Penicillium expansum (Ascomycota, Pezizomycotina, Eurotium) is a blue mold fungus, a postharvest pathogen of fruits such as apples, and the causative agent of blue rot disease. Lin et al., 2015 (Angew Chem Int Ed Engl, 54:3004-7) elucidated the genes for the biosynthetic pathway of commenecins from L-tryptophan in P. expansum, including the cnsB decarboxylase gene described herein, which encodes a PLP-dependent tryptophan decarboxylase. The amino acid sequences of the CnsB polypeptides from P. expansum are Uniprot accession number A0A0A2IDH4, shown herein in SEQ ID NO:1 (and referred to herein as "CnsBv1"), and GenBank accession number AMQ76109.1, shown herein in SEQ ID NO:3 (and referred to herein as "CnsBv2"). The nucleotide sequences of the cnsB genes encoding CnsBv1 and CnsB2 are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.

[0154] We also identified an active truncated form of CnsBv2 (referred to herein as "ΔCnsBv2") lacking 28 amino acid residues from the N-terminus of CnsBv2. The amino acid sequence of ΔCnsBv2 is shown in SEQ ID NO:5, and the encoding nucleotide sequence is shown in SEQ ID NO:6. CnsBv1 and CnsBv2 share 81% sequence identity over the entire length of their amino acid sequences (Figure 2A), while CnsBv1 and ΔCnsBv2 share 86% sequence identity (Figure 2B).

[0155] Additionally, the present inventors have identified a PLP-dependent tryptophan decarboxylase (referred to herein as "AsTDC1") from Aspergillus steinii (Ascomycota, Subphylum Acanthophylum, Class Eurotium). The amino acid sequence of the AsTDC1 polypeptide is shown in SEQ ID NO:7, and the encoding nucleotide sequence is shown in SEQ ID NO:8. asTDC1 shares 60.9% sequence identity with CnsBv1 and 62.1% sequence identity with CnsBv2 over the entire length of their amino acid sequences (see Figure 5).

[0156] Additionally, the present inventors have identified a PLP-dependent tryptophan decarboxylase (referred to herein as "CrTDC") from Catharanthus roseus (Dicotyledonous Plants). The amino acid sequence of the CrTDC polypeptide is set forth in SEQ ID NO:29, and the encoding nucleotide sequence is set forth in SEQ ID NO:30.

[0157] Additionally, the present inventors have identified a PLP-dependent tryptophan decarboxylase (referred to herein as "AcTDC") from Aspergillus ceratus (Ascomycota, Subphylum Acanthogonum, Class Eurotium). The amino acid sequence of the AcTDC polypeptide is set forth in SEQ ID NO:48.

[0158] The inventors also identified various amino acid sequences that are conserved in the enzymatically active PLP-dependent tryptophan decarboxylases described herein (e.g., AsTDC1, AcTDC) and confirmed that these conserved sequences are also found in other PLP-dependent tryptophan decarboxylases (e.g., non-Eurotiomycete fungi, bacteria, and plants).

[0159] Accordingly, embodiments of the present disclosure relate to PLP-dependent tryptophan decarboxylases that include one or all of the conserved sequences described herein, and uses thereof.

[0160] Accordingly, embodiments of the present disclosure relate to fungal PLP-dependent tryptophan decarboxylases derived from members of the Eurotium fungi, and uses thereof. Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a fungal PLP-dependent tryptophan decarboxylases derived from members of the Eurotium fungi, and uses thereof. Aspects and embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that include one or more conservative amino acid substitutions relative to a fungal PLP-dependent tryptophan decarboxylases derived from members of the Eurotium fungi, and uses thereof.

[0161] Fungi of the Eurotium fungi can belong to a subclass selected from, for example, Eurotiomycetidae, Mycocaliciomycetidae, Coryneliomycetidae, Sclerococcomycetidae, Cryptocaliciomycetidae, and Chaetothyriomycetidae. In exemplary embodiments, the fungal PLP-dependent tryptophan decarboxylase of the present disclosure is derived from a member of the Eurotium fungi.

[0162] Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases derived from members of the Ascomycota and / or Basidiomycota phyla, and uses thereof. Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a PLP-dependent tryptophan decarboxylases derived from members of the Ascomycota and / or Basidiomycota, and uses thereof. Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that include one or more conservative amino acid substitutions relative to a PLP-dependent tryptophan decarboxylases derived from members of the Ascomycota and / or Basidiomycota, and uses thereof.

[0163] Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases derived from members of the class Dicotyledonous Plants, and uses thereof. Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a PLP-dependent tryptophan decarboxylases derived from members of the class Dicotyledonous Plants, and uses thereof. Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that include one or more conservative amino acid substitutions relative to a PLP-dependent tryptophan decarboxylases derived from members of the class Dicotyledonous Plants, and uses thereof.

[0164] Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases derived from bacteria and uses thereof. Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a PLP-dependent tryptophan decarboxylases derived from bacteria, and uses thereof. Embodiments of the present disclosure also relate to PLP-dependent tryptophan decarboxylases that contain one or more conservative amino acid substitutions relative to a PLP-dependent tryptophan decarboxylases derived from bacteria, and uses thereof.

[0165] The present inventors surprisingly found that modifying the yeast S. cerevisiae to express CnsBv1 or AsTDC1 significantly increased tryptamine expression compared to S. cerevisiae strains expressing PsiD from Torpedo mushroom (PcPsiD; SEQ ID NO: 9). The PLP-dependent tryptophan decarboxylases of the present disclosure are only very distantly related to the non-PLP-dependent PcPsiD tryptophan decarboxylases. CnsBv1 and PcPsiD share only 16% sequence identity, CnsBv2 and PcPsiD share only 16.2% sequence identity, and ΔCnsBv2 and PcPsiD share only 15.3% sequence identity (see Figure 3). Similarly, AsTDC1 and PcPsiD share only 14% sequence identity (see Figure 5). The fungal PLP-dependent tryptophan decarboxylases of the present disclosure are also only distantly related to plant PLP-dependent tryptophan decarboxylases such as CrTDC. CnsBv1 and CnsBv2 share only 24.3% and 23.5% sequence identity with CrTDC, respectively (Figure 4). AsTDC1 and CrTDC1 share only 25% sequence identity (Figure 5).

[0166] The present inventors also surprisingly found that engineering the bacterial cell Escherichia coli to express AsTDC1 significantly increased tryptamine expression when compared to an E. coli strain expressing PsiD from S. pulcherrimus (Figure 15). The PLP-dependent tryptophan decarboxylases of the present disclosure are only very distantly related to the non-PLP-dependent PcPsiD tryptophan decarboxylases.

[0167] The present disclosure provides a cell containing an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a fungal pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase derived from a member of the Eurotium fungi class, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to the fungal PLP-dependent tryptophan decarboxylase or contains one or more conservative amino acid substitutions thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not possess the exogenous gene and contains a non-PLP-dependent tryptophan decarboxylase, optionally PsiD. The cell can contain one or more copies of a gene encoding the PLP-dependent tryptophan decarboxylase. Optionally, the equivalent cell contains a PsiD tryptophan decarboxylase having the amino acid sequence of SEQ ID NO:9. Optionally, the at least one tryptophan derivative whose production is increased in the cells of the disclosure is tryptamine or psilocybin.

[0168] The present disclosure also provides a cell containing an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a sequence having at least about 70% sequence identity thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not possess the exogenous gene and contains a non-PLP-dependent tryptophan decarboxylase, optionally PsiD. The cell can contain one or more copies of a gene encoding the PLP-dependent tryptophan decarboxylase. Optionally, the equivalent cell contains a PsiD tryptophan decarboxylase having the amino acid sequence of SEQ ID NO:9. Optionally, the at least one tryptophan derivative whose production is increased in the cell of the present disclosure is tryptamine or psilocybin.

[0169] The present disclosure additionally provides a cell comprising an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not possess the exogenous gene and includes a non-PLP-dependent tryptophan decarboxylase, optionally PsiD. The cell can include one or more copies of a gene encoding the PLP-dependent tryptophan decarboxylase. Optionally, the equivalent cell includes a PsiD tryptophan decarboxylase having the amino acid sequence of SEQ ID NO:9. Optionally, the at least one tryptophan derivative whose production is increased in the cell of the present disclosure is tryptamine or psilocybin.

[0170] The present disclosure also provides a cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding a pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that either lacks the exogenous polynucleotide or comprises a second polynucleotide encoding a second tryptophan decarboxylase, and the second polynucleotide is not the same as the exogenous polynucleotide.

[0171] The present disclosure also provides a cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding a pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that either lacks the exogenous polynucleotide or comprises a second polynucleotide encoding a second tryptophan decarboxylase, wherein the second polynucleotide is not the same as the exogenous polynucleotide, and the PLP-dependent tryptophan decarboxylase comprises one or more or all of the following:

[0172] a) GX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 A first amino acid sequence of R, wherein: X1, X2, X3, X4, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , and X 16 is any amino acid residue, X5 and X6 are amino acid residues selected from the group consisting of glycine, alanine, serine, and threonine; X7 is an amino acid residue selected from the group consisting of serine and threonine, and X 17 is an amino acid residue selected from the group consisting of alanine and glycine;

[0173] b) X1X2X3X4X5X6HX7X8X9X 10 KX 11 X 12 X 13 X 14 X 15 X 16 a second amino acid sequence of: X1 is any amino acid residue selected from the group consisting of tyrosine, leucine, phenylalanine, valine, cysteine, and tryptophan; X2, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 and X 15 is any amino acid residue, X3 is any amino acid residue selected from the group consisting of serine and threonine; and X 16 is an amino acid residue selected from the group consisting of glycine, alanine, serine and threonine;

[0174] c) DX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 GX 15 X 16 X 17 X 18 X 19 X 20 X 21 A third amino acid sequence of D, wherein: X1, X2, X3, X4, X5, X6, X8, X9, X 10 , X11 , X 12 , X 13 , X 14 , X 15 , X 17 , X 18 , X 19 and X 21 is any amino acid residue, and X7 is an amino acid residue selected from the group consisting of proline and asparagine; X 16 is an amino acid residue selected from the group consisting of threonine, proline, and valine, and X 20 is 1, 2, 3, 4, 5, or 6 amino acid residues, and is any amino acid residue;

[0175] d)X1X2HX3X4X5X6X7X8X9X 10 a fourth amino acid sequence, wherein: X1 is an amino acid residue selected from the group consisting of tryptophan and phenylalanine; X2, X3, X6, X9 and X 10 is any amino acid residue, X4 is an amino acid residue selected from the group consisting of aspartic acid and glutamic acid; X5 is an amino acid residue selected from the group consisting of glycine, alanine, and serine; X7 is an amino acid residue selected from the group consisting of phenylalanine, tyrosine and tryptophan, and X8 is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; and

[0176] e)X1X2X3X4X5X6X7HKX8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 a fifth amino acid sequence of: X1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, histidine, and asparagine; X2 is an amino acid residue selected from the group consisting of serine and threonine; X3, X4, X5, X7, X 10 , X 11 , X 12 , X 13 and X 14 is any amino acid residue, X6 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, and threonine; X8 is an amino acid residue selected from the group consisting of tryptophan, phenylalanine, tyrosine, leucine, methionine, and cysteine; X9 is an amino acid residue selected from the group consisting of leucine, isoleucine, valine, methionine and phenylalanine; X 15 is an amino acid residue selected from the group consisting of cysteine, alanine and serine, and X 16 is an amino acid residue selected from the group consisting of glycine, serine, alanine and threonine.

[0177] The present disclosure also provides a cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that lacks the exogenous polynucleotide or that includes a second polynucleotide encoding a second tryptophan decarboxylase, and the second polynucleotide is not the same as the exogenous polynucleotide.

[0178] As used herein, the term "equivalent cell" refers to a cell of the same type, origin, source, and composition (i.e., the same genome, protein and carbohydrate composition, etc.) as a cell of the present disclosure, except that the cell does not contain an exogenous gene encoding a PLP-dependent tryptophan carboxylase of the present disclosure and optionally contains a non-PLP-dependent, PsiD tryptophan decarboxylase. Thus, "equivalent cell" is used for comparison purposes, specifically for determining the production level of one or more tryptophan derivatives in the presence of a PLP-dependent tryptophan carboxylase of the present disclosure and a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from Eurotium or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto, optionally a PsiD tryptophan decarboxylase. Typically, "equivalent cells" to cells of the present disclosure are maintained, preserved, and cultured under the same conditions.

[0179] In accordance with the present disclosure, the cell can be a eukaryotic cell, a bacterial cell, or a microalgae cell, typically a eukaryotic cell. In certain embodiments, the cell is not from the species from which the original amino acid sequence or polynucleotide is derived (e.g., the cell is heterologous to the amino acid sequence or polynucleotide). In certain embodiments, the cell is not a P. expansum cell or an A. steinii cell. In some embodiments, the cell is not an A. seratus cell.

[0180] In exemplary embodiments, the cell is a yeast cell. Yeast cells include, for example, cells of the genera Saccharomyces, Yarrowia, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Candida, Cryptococcus (such as C. aerius), Pichia (also known as Komagataella), Debaromyces, Zygosaccharomyces (such as Z. baileyi), Torulaspora ( Torulaspora (such as T. delbrueckii), Brettanomyces (such as B. bruxellensis), Penicillium, Rhizopus, Fusarium, Fusdium, Hansenula, Gibberella, Mucor, Mortierella, or Trichoderma. In certain exemplary embodiments, the yeast is a Saccharomyces species, such as S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, or S. boulardii. In another exemplary embodiment, the yeast is a Yarrowia species, such as Y. lipolytica. In another exemplary embodiment, the yeast is a Kluyveromyces species, such as K. dogzhanskii, K. lactis, K. marxianus var. marxianus, or K. thermotolerans.In another exemplary embodiment, the yeast is a species of the genus Schizosaccharomyces, such as S. pombe, S. japonicus, or S. cryophilus. In another exemplary embodiment, the yeast is a species of the genus Pichia (Komagataella), such as P. pastoris, P. stipidis, P. sorbitophila, or K. phaffi.

[0181] In exemplary embodiments, the cell is a filamentous fungal cell. The filamentous fungus may be, for example, an Aspergillus species, such as A. nidulans, A. oryzae, A. niger, or A. terreus. The filamentous fungus may be, for example, a Penicillium species, such as P. chrysogenum, P. rubens, P. expansum, or P. paxilli.

[0182] Alternatively, the eukaryotic cell may be a plant cell. By way of non-limiting example only, the plant may be selected from species of the genus Arabidopsis, such as Arabidopsis thaliana (A. thaliana), species of the genus Zea, such as Z. mays, species of the genus Medicago, such as M. truncatula, species of the genus Nicotiana, such as N. tabacum, or species of the genus Glycine, such as Glycine max (Glycine max).

[0183] Alternatively, the cell may be a bacterial cell. By way of non-limiting example only, the bacterium may be selected from species belonging to the genus Bacillus, such as B. subtilis, species belonging to the genus Escherichia, such as E. coli, species belonging to the genus Lactobacillus, such as L. casei, species belonging to the genus Lactococcus, such as L. lactis, species belonging to the genus Corynebacterium, such as C. glutamicum, species belonging to the genus Acetobacter, species belonging to the genus Acinetobacter, species belonging to the genus Pseudomonas, such as P. putida, or species belonging to the genus Streptomyces, such as S. coelicolor.

[0184] The PLP-dependent tryptophan decarboxylase encoded by the exogenous gene can comprise the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the tryptophan decarboxylase sequence set forth in SEQ ID NO: 1. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising the amino acid sequence set forth in SEQ ID NO: 1. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising an amino acid sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:1.

[0185] The PLP-dependent tryptophan decarboxylase encoded by the exogenous gene can comprise the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the tryptophan decarboxylase sequence set forth in SEQ ID NO: 3. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising the amino acid sequence set forth in SEQ ID NO: 3. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising an amino acid sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:3.

[0186] The PLP-dependent tryptophan decarboxylase encoded by the exogenous gene can comprise the amino acid sequence set forth in SEQ ID NO: 5, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the tryptophan decarboxylase sequence set forth in SEQ ID NO: 5. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising the amino acid sequence set forth in SEQ ID NO: 5. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising an amino acid sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:5.

[0187] The PLP-dependent tryptophan decarboxylase encoded by the exogenous gene can comprise the amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the tryptophan decarboxylase sequence set forth in SEQ ID NO: 7. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising the amino acid sequence set forth in SEQ ID NO: 7. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising an amino acid sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:7.

[0188] The PLP-dependent tryptophan decarboxylase encoded by the exogenous gene can comprise the amino acid sequence set forth in SEQ ID NO: 29, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the tryptophan decarboxylase sequence set forth in SEQ ID NO: 29. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising the amino acid sequence set forth in SEQ ID NO: 29. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising an amino acid sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:29.

[0189] The PLP-dependent tryptophan decarboxylase encoded by the exogenous gene can comprise the amino acid sequence set forth in SEQ ID NO: 48, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the tryptophan decarboxylase sequence set forth in SEQ ID NO: 48. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising the amino acid sequence set forth in SEQ ID NO: 48. Embodiments of the present disclosure provide a PLP-dependent tryptophan decarboxylase comprising an amino acid sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:48.

[0190] The exogenous gene encoding a PLP-dependent tryptophan decarboxylase can comprise the nucleic acid sequence set forth in SEQ ID NO: 2, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 2, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 2 or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by the nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:2, or a nucleic acid sequence complementary thereto.

[0191] The exogenous gene encoding a PLP-dependent tryptophan decarboxylase can comprise the nucleic acid sequence set forth in SEQ ID NO: 4, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 4, or a complementary nucleic acid sequence thereto. Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 4, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 4 or a complementary nucleic acid sequence thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by the nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 4, or a nucleic acid sequence complementary thereto.

[0192] The exogenous gene encoding a PLP-dependent tryptophan decarboxylase can comprise the nucleic acid sequence set forth in SEQ ID NO: 6, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 6, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 6 or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by the nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:6, or a nucleic acid sequence complementary thereto.

[0193] The exogenous gene encoding a PLP-dependent tryptophan decarboxylase can comprise the nucleic acid sequence set forth in SEQ ID NO: 8, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 8, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 8 or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by the nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO:8, or a nucleic acid sequence complementary thereto.

[0194] The exogenous gene encoding a PLP-dependent tryptophan decarboxylase can comprise the nucleic acid sequence set forth in SEQ ID NO: 30, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, or a complementary nucleic acid sequence thereto. Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 30, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 30 or a complementary nucleic acid sequence thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by the nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 30, or a nucleic acid sequence complementary thereto.

[0195] The exogenous gene encoding a PLP-dependent tryptophan decarboxylase can comprise the nucleic acid sequence set forth in SEQ ID NO: 49, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, or a complementary nucleic acid sequence thereto. Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 49, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 49 or a complementary nucleic acid sequence thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by the nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 49, or a nucleic acid sequence complementary thereto.

[0196] The coding sequence of the PLP-dependent tryptophan decarboxylase gene can be codon-optimized for expression in a selected host cell. Methods for codon-optimizing coding sequences are well known to those skilled in the art.

[0197] By way of example, the coding sequence of a PLP-dependent tryptophan decarboxylase gene can be codon-optimized for expression in S. cerevisiae and can include the nucleic acid sequence set forth in SEQ ID NO:23, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:23, or a nucleic acid sequence complementary thereto.

[0198] In another example, the coding sequence of the codon-optimized PLP-dependent tryptophan decarboxylase gene can include the nucleic acid sequence set forth in SEQ ID NO: 31, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 31, or a nucleic acid sequence complementary thereto.

[0199] Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 23, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 23, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences and PLP-dependent tryptophan decarboxylases encoded by nucleic acid sequences having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 23, or a nucleic acid sequence complementary thereto.

[0200] Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 31, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 31, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences and PLP-dependent tryptophan decarboxylases encoded by nucleic acid sequences having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 31, or a nucleic acid sequence complementary thereto.

[0201] In one example, the coding sequence of the codon-optimized PLP-dependent tryptophan decarboxylase gene can include the nucleic acid sequence set forth in SEQ ID NO: 47, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 47, or a nucleic acid sequence complementary thereto.

[0202] Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 47, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 47, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 47, or a nucleic acid sequence complementary thereto.

[0203] In one example, the coding sequence of the codon-optimized PLP-dependent tryptophan decarboxylase gene can include the nucleic acid sequence set forth in SEQ ID NO: 50, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 50, or a nucleic acid sequence complementary thereto.

[0204] Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 50, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 50, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences, and PLP-dependent tryptophan decarboxylases encoded by nucleic acid sequences, having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 50, or a nucleic acid sequence complementary thereto.

[0205] In one example, the coding sequence of the codon-optimized PLP-dependent tryptophan decarboxylase gene can include the nucleic acid sequence set forth in SEQ ID NO: 51, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 51, or a nucleic acid sequence complementary thereto.

[0206] Embodiments of the present disclosure provide an isolated nucleotide sequence set forth in SEQ ID NO: 51, and a PLP-dependent tryptophan decarboxylase encoded by the nucleic acid sequence set forth in SEQ ID NO: 51, or a nucleic acid sequence complementary thereto. Embodiments of the present disclosure provide isolated nucleotide sequences and PLP-dependent tryptophan decarboxylases encoded by nucleic acid sequences having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 51, or a nucleic acid sequence complementary thereto.

[0207] In certain embodiments of the present disclosure, the exogenous gene encoding a PLP-dependent tryptophan decarboxylase is operably linked to a promoter and / or other regulatory elements. Typically, the promoter and / or other regulatory elements are not naturally associated with (i.e., are heterologous to) the gene encoding the PLP-dependent tryptophan decarboxylase of the present disclosure. The promoter can be a constitutive promoter or an inducible promoter. Examples of constitutive promoters useful in yeast cells include, but are not limited to, the PGK (phosphoglycerate kinase) promoter, the ADH-1 (alcohol dehydrogenase) promoter, the ENO (enolase) promoter, the glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter (also referred to as the TDH3 promoter), the constitutive cell wall (CCW) promoter, the histone (HHF) promoter, the hexose transporter (HXT) promoter, the PEP carboxykinase (PCK) promoter, the PYK-1 (pyruvate kinase) promoter, the translation elongation factor-1-alpha (TEF) promoter, and the CYC-1 (cytochrome c oxidase promoter) promoter. In one embodiment, the yeast promoter is a S. cerevisiae promoter. Exemplary constitutive promoters include, but are not limited to, pTDH3, pCCW12, pPGK1, pTEF1, pHHF2, pHXT7, pTEF2, pHHF1, pADH2, pPCK1, pMLS1, pICL1, and pPHO89.

[0208] In another embodiment, the constitutive promoter may not be derived from yeast. Examples of such promoters include, but are not limited to, the cauliflower mosaic virus 35S promoter, the glucocorticoid response element, and the androgen response element. A constitutive promoter may be a naturally occurring molecule or a variant thereof containing, for example, one, two, or three nucleotide substitutions that do not abolish (and preferably enhance) promoter function.

[0209] Effective conditions for culturing the cells of the present disclosure are known to those skilled in the art and include, but are not limited to, suitable media, bioreactors, temperature, pH, and oxygen conditions that permit secondary metabolite production, and in particular the production of tryptophan derivatives. By suitable media, we mean any medium in which cells can be cultured to produce the tryptophan derivatives defined herein. Such media typically comprise an aqueous medium having assimilable carbon, nitrogen, and phosphate sources, as well as other nutrients such as appropriate salts, minerals, metals, and vitamins. The cells defined herein may be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and Petri plates. Suitable culture conditions are within the expertise of those skilled in the art.

[0210] Tryptophan derivatives Embodiments of the present disclosure provide cells and methods for the biosynthetic production of one or more tryptophan derivatives, particularly tryptamine-derived compounds, such as tryptamine and tryptamine-derived alkaloids. The cnsB, asTDC1, AcTDC, and CrTDC genes described herein encode PLP-dependent tryptophan decarboxylases that catalyze the decarboxylation of aliphatic carboxylic acids, converting L-tryptophan to tryptamine. Accordingly, certain embodiments of the present disclosure provide cells and methods for the production of tryptamine.

[0211] Cells of the present disclosure that include an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure can further include one or more additional genes (which can be endogenous or exogenous to the host cell) that encode products, typically enzymes, required for the biosynthetic production of one or more tryptamine-derived compounds. Such compounds include, but are not limited to, 4-hydroxytryptamine, norbaeocystin, baeocystin, psilocybin, psilocin, norpsilocin, N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenin), N,N,N-trimethyltryptamine, N-methyltryptamine, serotonin (5-hydroxytryptamine), N-acetylserotonin, melatonin, aeruginacins, communesins (such as communesin A, communesin B, and communesin F), harmala alkaloids (such as deoxyvasicine, deoxyvasicinone, harmine, harmaline, tetrahydroharmine, and vasicine), ajmalicine, ajmaline, yohimbine, quinine, vincristine, vinblastine, and camptothecin. In exemplary embodiments, the cells of the present disclosure are capable of producing one or more of 4-hydroxytryptamine, norbeocystin, psilocybin, and psilocin. Those skilled in the art will appreciate that such compounds can also include halogenated, fluorinated, deuterated, and / or substituted versions or fragments thereof.

[0212] In addition to containing an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, the cells of the present disclosure can further contain a gene encoding a tryptamine 4-monooxygenase, such as PsiH, typically for the production of 4-hydroxytryptamine. The cells of the present disclosure can contain one or more copies (e.g., 1, 2, 3, 4, or more copies) of a tryptamine 4-monooxygenase gene, which can be endogenous to the cell or can be provided exogenously, for example, in a vector of the present disclosure. The tryptamine 4-monooxygenase can comprise the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 11. The coding sequence of the tryptamine 4-monooxygenase gene can be codon-optimized for expression in a selected host cell, such as S. cerevisiae (as set forth in SEQ ID NO: 25). Methods for codon optimization of coding sequences are well known to those skilled in the art. The tryptamine 4-monooxygenase enzyme can be encoded by a nucleic acid sequence set forth in SEQ ID NO: 12 or 25, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12 or 25.Optionally, the PsiH tryptamine 4-monooxygenase is derived from Psilocybe cyanescens (SEQ ID NO: 11); however, numerous other sources of PsiH can be utilized, including, for example, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus junonius, and Gymnopilus dilepsis, as would be known to one of skill in the art.

[0213] In addition to containing an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, the cells of the present disclosure can further contain a gene encoding a 4-hydroxytryptamine kinase, such as PsiK, typically for the production of norbaeocystin. The cells of the present disclosure can contain one or more copies (e.g., 1, 2, 3, 4, or more copies) of a 4-hydroxytryptamine kinase gene, which can be endogenous to the cell or can be provided exogenously, for example, in a vector of the present disclosure. The 4-hydroxytryptamine kinase can comprise the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13. The coding sequence of the 4-hydroxytryptamine kinase gene can be codon-optimized for expression in a selected host cell, such as S. cerevisiae (as set forth in SEQ ID NO: 26). Methods for codon optimization of coding sequences are well known to those skilled in the art. The 4-hydroxytryptamine kinase enzyme can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 14 or 26, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14 or 26. Optionally, the PsiK 4-hydroxytryptamine kinase is derived from PsiKiae (SEQ ID NO: 11); however, numerous other sources of PsiK can be utilized, including, for example, Psilocybe kyanense, Psammophila fascicularis, Psammophila spp., and Psammophila purpurea, as will be known to those of skill in the art.

[0214] In addition to containing an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, the cells of the present disclosure can further contain a gene encoding a methyltransferase (psilocybin synthase), such as PsiM, typically for the production of psilocybin. The cells of the present disclosure can contain one or more copies (e.g., 1, 2, 3, 4, or more copies) of the psilocybin synthase gene, which can be endogenous to the cell or can be provided exogenously, for example, in a vector of the present disclosure. The psilocybin synthase can comprise the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 15. The coding sequence of the psilocybin synthase gene can be codon-optimized for expression in a selected host cell, such as S. cerevisiae (as set forth in SEQ ID NO: 27). Methods for codon optimization of coding sequences are well known to those skilled in the art. The psilocybin synthase enzyme can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 16 or 27, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16 or 27. Optionally, the PsiM psilocybin synthase is derived from PsiMella punctata (SEQ ID NO: 15); however, numerous other sources of PsiM can be utilized, including, for example, Psilocybe kianescens, Psammophila nigricans, Psammophila spp., and Psammophila purpurea, as will be known to those of skill in the art.

[0215] In addition to containing an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, the cells of the present disclosure can further contain a gene encoding a methyltransferase, such as RmNMT. The cells of the present disclosure can contain one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding an RmNMT, which can be endogenous to the cell or can be provided exogenously, for example, in a vector of the present disclosure. RmNMT can comprise the amino acid sequence set forth in SEQ ID NO: 42, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 42. The coding sequence of RmNMT can be codon-optimized for expression in a selected host cell, such as S. cerevisiae (as set forth in SEQ ID NO: 44). Methods for codon optimization of coding sequences are well known to those skilled in the art. The RmNMT can be encoded by a nucleic acid sequence set forth in SEQ ID NO: 43 or 44, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 43 or 44.

[0216] For the production of psilocin, the cells of the present disclosure, in addition to containing an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, can further contain a gene encoding a 4-hydroxytryptamine kinase, such as PsiK, as described above. PsiK can comprise the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 13. The PsiK enzyme can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 14 or 26, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14 or 26. Optionally, PsiK is derived from Drosophila chinensis (SEQ ID NO: 11); however, numerous other sources of PsiK can be utilized, including, for example, Psilocybe kianescens, Psammophila nigricans, Psammophila spp., and Psammophila purpurea, as will be known to those of skill in the art.

[0217] In addition to containing an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, the cells of the present disclosure can further contain a gene encoding a cytochrome P450 reductase, such as Cpr from Torpedo. The cells of the present disclosure can contain one or more copies (e.g., 1, 2, 3, 4 or more copies) of a cytochrome P450 reductase gene, which can be endogenous to the cell or can be provided exogenously, for example, in a vector of the present disclosure. The cytochrome P450 reductase can comprise the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 17. The coding sequence of the cytochrome P450 reductase gene can be codon-optimized for expression in a selected host cell, such as S. cerevisiae (as set forth in SEQ ID NO: 28). Methods for codon optimization of coding sequences are well known to those skilled in the art. The cytochrome P450 reductase can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 18 or 28, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 18 or 28.

[0218] In addition to containing an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, the cells of the present disclosure can further contain a gene encoding a cytochrome P450 reductase, such as OsCPR from rice (Oryza sativa Japonica Group). The cells of the present disclosure can contain one or more copies (e.g., 1, 2, 3, 4, or more copies) of OsCPR, which can be endogenous to the cell or can be provided exogenously, for example, in a vector of the present disclosure. OsCPR can comprise the amino acid sequence set forth in SEQ ID NO: 39, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 39. The coding sequence of OsCPR can be codon-optimized for expression in a selected host cell, such as S. cerevisiae (as set forth in SEQ ID NO: 41). Methods for codon optimization of coding sequences are well known to those skilled in the art. The OsCPR can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 40 or 41, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 40 or 41.

[0219] In certain exemplary embodiments, the cells of the present disclosure contain one or more copies (e.g., 1, 2, 3, 4 or more copies) of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto; one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a tryptamine 4-monooxygenase (e.g., PsiH) having the amino acid sequence set forth in SEQ ID NO:11, or a sequence having at least about 70% sequence identity thereto; The cell comprises one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a 4-hydroxytryptamine kinase (e.g., PsiK) having the amino acid sequence set forth in SEQ ID NO: 15 or a sequence having at least about 70% sequence identity thereto, one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a methyltransferase (psilocybin synthase) (e.g., PsiM) having the amino acid sequence set forth in SEQ ID NO: 15 or a sequence having at least about 70% sequence identity thereto, and optionally one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a cytochrome 450 reductase (e.g., Cpr) having the amino acid sequence set forth in SEQ ID NO: 17 or a sequence having at least about 70% sequence identity thereto. Typically, the cell is capable of producing psilocybin and / or psilocin.

[0220] In certain embodiments, the cells of the present disclosure contain one or more copies (e.g., 1, 2, 3, 4 or more copies) of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto; one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a tryptamine 4-monooxygenase (e.g., PsiH) having the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least about 70% sequence identity thereto; one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding an amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least about 70% sequence identity thereto; one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a 4-hydroxytryptamine kinase (e.g., PsiK) having the amino acid sequence set forth in SEQ ID NO: 15 or a sequence having at least about 70% sequence identity thereto, one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a methyltransferase (psilocybin synthase) (e.g., PsiM) having the amino acid sequence set forth in SEQ ID NO: 15 or a sequence having at least about 70% sequence identity thereto, and optionally one or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a cytochrome 450 reductase (e.g., Cpr) having the amino acid sequence set forth in SEQ ID NO: 17 or a sequence having at least about 70% sequence identity thereto.

[0221] In certain embodiments, the cells of the present disclosure contain one or more copies (e.g., 1, 2, 3, 4 or more copies) of an exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto, one or more copies (e.g., 1, 2, 3, 4 or more copies) of an exogenous polynucleotide encoding a methyltransferase (e.g., RmNMT) having the amino acid sequence set forth in SEQ ID NO: 42, or a sequence having at least about 70% sequence identity thereto. , 2, 3, 4 or more copies) of a gene encoding a cytochrome P450 reductase (e.g., OsCPR) having the amino acid sequence set forth in SEQ ID NO: 39 or a sequence having at least about 70% sequence identity thereto; and 1 or 2 or more copies (e.g., 1, 2, 3, 4 or more copies) of a gene encoding a methyltransferase (e.g., OsT5H) having the amino acid sequence set forth in SEQ ID NO: 36 or a sequence having at least about 70% sequence identity thereto.

[0222] In exemplary embodiments, the cells of the present disclosure contain one copy of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto. The cells can also contain one or more copies of one or more additional tryptophan decarboxylase genes, including, for example, psiD and / or crTDC. The tryptophan decarboxylase genes can be provided to the cells on the same or different vectors.

[0223] In embodiments, the cells of the present disclosure comprise one copy of an exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto. The cells can also comprise one or more copies of one or more additional tryptophan decarboxylase genes, including, for example, psiD, AcTDC, AsTDC1, and / or crTDC. The tryptophan decarboxylase genes can be provided to the cells on the same or different vectors.

[0224] In an exemplary embodiment, the cell of the present disclosure contains two copies of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto. The cell can also contain one or more copies of one or more additional tryptophan decarboxylase genes, including, for example, psiD and / or crTDC. The tryptophan decarboxylase genes can be provided to the cell on the same or different vectors. In an exemplary embodiment, the cell further contains two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene, two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene, two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene, and two copies of a cytochrome 450 reductase (e.g., Cpr) gene. In an exemplary embodiment, a cell of the present disclosure comprises three copies of a methyltransferase (psilocybin synthase) (eg, PsiM) gene.

[0225] In an embodiment, the cell of the present disclosure contains two copies of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto. The cell can also contain one or more copies of one or more additional tryptophan decarboxylase genes, including, for example, psiD, AcTDC, AsTDC1, and / or crTDC. The tryptophan decarboxylase genes can be provided to the cell on the same or different vectors. In an exemplary embodiment, the cell further contains two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene, two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene, two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene, and two copies of a cytochrome 450 reductase (e.g., Cpr) gene. In an exemplary embodiment, a cell of the present disclosure comprises three copies of a methyltransferase (psilocybin synthase) (eg, PsiM) gene.

[0226] The yeast adenosylhomocysteinase (Sah1) and adenosine kinase (Ado1) enzymes have been reported to recycle the cofactors S-adenosylmethionine (SAM) and S-adenosylhomocysteine ​​(SAH) and increase PsiM methyltransferase activity. Thus, cells of the present disclosure can include one or more copies (e.g., two, three, four, or more copies) of a gene encoding Sah1 and / or a gene encoding Ado1. The gene encoding Sah1 can be the S. cerevisiae SAH1 gene nucleic acid sequence set forth in SEQ ID NO:20, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:20. The Sah1 enzyme can comprise the amino acid sequence set forth in SEQ ID NO:19, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:19. The gene encoding Ado1 can be the S. cerevisiae ADO1 gene nucleic acid sequence set forth in SEQ ID NO:22, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:22.The Ado1 enzyme can comprise the amino acid sequence set forth in SEQ ID NO:21, or a sequence having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:21.

[0227] In exemplary embodiments, a cell of the present disclosure comprises at least two copies of the SAH1 gene and at least two copies of the ADO1 gene. One or more copies of each gene can be endogenous to the cell. One or more copies of each gene can be exogenous to the cell. For example, in embodiments of the present disclosure in which the cell is S. cerevisiae, the cell comprises the SAH1 and ADO1 genes and is also provided with additional copies of the SAH1 and ADO1 genes.

[0228] In an exemplary embodiment, a cell of the present disclosure comprises one copy of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto, two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene, two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene, two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene, two copies of a cytochrome 450 reductase (e.g., Cpr) gene, one exogenously supplied copy of a SAH1 gene, and one exogenously supplied copy of an ADO1 gene.

[0229] In an exemplary embodiment, a cell of the present disclosure comprises two copies of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto, two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene, two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene, two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene, two copies of a cytochrome 450 reductase (e.g., Cpr) gene, one exogenously supplied copy of a SAH1 gene, and one exogenously supplied copy of a ADO1 gene.

[0230] In an exemplary embodiment, a cell of the present disclosure comprises one copy of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto; one copy of a gene encoding an additional tryptophan decarboxylase (e.g., PsiD or CrTDC); two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene; two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene; two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene; two copies of a cytochrome 450 reductase (e.g., Cpr) gene; one exogenously supplied copy of a SAH1 gene; and one exogenously supplied copy of an ADO1 gene.

[0231] In an embodiment, a cell of the present disclosure comprises one copy of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto, two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene, two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene, two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene, two copies of a cytochrome 450 reductase (e.g., Cpr) gene, one exogenously supplied copy of a SAH1 gene, and one exogenously supplied copy of an ADO1 gene.

[0232] In an embodiment, a cell of the present disclosure comprises two copies of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto, two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene, two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene, two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene, two copies of a cytochrome 450 reductase (e.g., Cpr) gene, one exogenously supplied copy of a SAH1 gene, and one exogenously supplied copy of an ADO1 gene.

[0233] In embodiments, the cells of the present disclosure comprise one copy of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto; one copy of a gene encoding an additional tryptophan decarboxylase (e.g., PsiD or CrTDC); two copies of a tryptamine 4-monooxygenase (e.g., PsiH) gene; two copies of a 4-hydroxytryptamine kinase (e.g., PsiK) gene; two or three copies of a methyltransferase (psilocybin synthase) (e.g., PsiM) gene; two copies of a cytochrome 450 reductase (e.g., Cpr) gene; one exogenously supplied copy of a SAH1 gene; and one exogenously supplied copy of an ADO1 gene.

[0234] The present disclosure provides methods for the biosynthetic production of one or more tryptophan derivatives, particularly tryptamine-derived compounds such as tryptamine and tryptamine-derived alkaloids, as described herein. The methods include culturing a cell of the present disclosure under conditions suitable for promoting the production of one or more tryptophan derivatives. It should be understood that conditions suitable for promoting the production of one or more tryptophan derivatives also encompass conditions suitable for the expression of one or more of the genes described herein that are required for the production of one or more tryptophan derivatives. Such conditions will be well known to those of skill in the art.

[0235] Because the cells and methods of the present disclosure relate to the biosynthesis of tryptophan derivatives, it may be desirable in certain embodiments to increase the production of L-tryptophan in the cells of the present disclosure. For example, the cells of the present disclosure can overexpress one or more genes responsible for L-tryptophan production in S. cerevisiae, such as TRP1, TRP2, TRP3, TRP4, or TRP5, or can include one or more mutations in such genes that promote increased production and therefore availability of tryptophan. Alternatively, or in addition, the cells of the present disclosure can include one or more mutations in genes encoding transcriptional repressors of the aromatic amino acid precursor pathway in S. cerevisiae, such as ARO1, ARO2, ARO3, and / or ARO4, to increase the production and therefore availability of tryptophan.

[0236] Effective conditions for culturing the cells of the present disclosure are known to those skilled in the art and include, but are not limited to, suitable media, bioreactors, temperature, pH, and oxygen conditions that permit secondary metabolite production, and in particular the production of tryptophan derivatives. By suitable media, we mean any medium in which cells can be cultured to produce the tryptophan derivatives defined herein. Such media typically comprise an aqueous medium having assimilable carbon, nitrogen, and phosphate sources, as well as other nutrients such as appropriate salts, minerals, metals, and vitamins. The cells defined herein may be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and Petri plates. Suitable culture conditions are within the expertise of those skilled in the art.

[0237] Also provided herein are methods for the extraction of one or more tryptophan derivatives produced according to the methods of the present disclosure. Extraction can include lysing the cells to release the compounds, allowing them to be purified or isolated by methods known to those of skill in the art. Alternatively, the compounds can be secreted by the cells into the culture medium, allowing them to be isolated or purified therefrom using methods known to those of skill in the art.

[0238] Cell-free production The present disclosure also provides methods for the cell-free production of one or more tryptophan derivatives, particularly tryptamine-derived compounds such as tryptamine and tryptamine-derived alkaloids, as described herein. The methods include contacting tryptophan with at least one pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase described herein under conditions suitable for promoting the production of one or more tryptophan derivatives. References to conditions suitable for promoting the production of one or more tryptophan derivatives should also be understood to encompass conditions suitable for decarboxylation, hydroxylation, phosphorylation, N-methylation, and / or phosphorylation required for the production of one or more tryptophan derivatives. Such conditions will be well known to those skilled in the art.

[0239] In some embodiments, the PLP-dependent tryptophan decarboxylase and / or tryptophan may be provided as a cell lysate or purified and / or isolated protein. Methods for isolating proteins and / or producing recombinant proteins are known in the art and are described, for example, in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T.A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991); D.M.G.lover and B.D. Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996); and F.M.A.usubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current revisions); Ed. Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988).

[0240] Effective conditions for the production of tryptophan derivatives are known to those skilled in the art and include, but are not limited to, suitable media, bioreactors, temperature, pH, and oxygen conditions that permit secondary metabolite production, and in particular the production of tryptophan derivatives. By suitable substrate is meant any substrate in which PLP-dependent tryptophan decarboxylase is known to produce tryptophan derivatives as defined herein. Suitable conditions are within the expertise of those skilled in the art.

[0241] Also provided herein are methods for the extraction of one or more tryptophan derivatives produced according to the methods of the present disclosure. Extraction can include lysing the cells to release the compounds, allowing them to be purified or isolated by methods known to those of skill in the art. Alternatively, the compounds can be secreted by the cells into the culture medium, allowing them to be isolated or purified therefrom using methods known to those of skill in the art.

[0242] Compositions and Uses Also provided herein are compositions comprising the cells of the present disclosure or one or more tryptophan derivatives extracted from the cells of the present disclosure. The compositions can also include additional components such as diluents, stabilizers, excipients, and adjuvants. Compositions comprising the cells of the present disclosure, for example, yeast cells, can be administered to a subject as probiotics.

[0243] In certain examples, pharmaceutical compositions are provided that include one or more tryptophan derivatives extracted from the cells of the present disclosure and a pharmaceutically acceptable carrier. Carriers, diluents, and adjuvants include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween™, Pluronics™, or polyethylene glycol (PEG). In some embodiments, the physiologically acceptable carrier is a pH-buffered aqueous solution.

[0244] The compositions of the present disclosure can be utilized in the treatment or prevention of diseases or disorders in which one or more tryptophan derivatives as described herein may have therapeutic benefit. Typically, the diseases or disorders are neurodevelopmental, neurological, neuromuscular, movement, psychiatric, or psychological diseases, disorders, or syndromes. Such diseases, disorders, and syndromes include, by way of non-limiting example only, depression, such as major depressive disorder or treatment-resistant depression, anxiety disorders, obsessive-compulsive disorders, personality disorders, substance addiction or dependence, such as alcohol or tobacco addiction or dependence, post-traumatic stress disorder, migraine, and chronic headache. Accordingly, provided herein are methods for treating or preventing diseases or disorders as described herein above, comprising administering a composition of the present disclosure to a subject. The subject may be suffering from, suspected of suffering from, or at risk of suffering from a disease or disorder, including depression or a depressive disorder, anxiety disorder, obsessive-compulsive disorder, personality disorder, substance addiction or dependence, post-traumatic stress disorder, migraine, and / or chronic headache.

[0245] Nucleotide sequences, nucleic acid constructs and vectors Provided herein is an isolated nucleotide sequence comprising a gene encoding a fungal PLP-dependent tryptophan decarboxylase from the class Eurotium. Optionally, the gene is operably linked to a heterologous promoter capable of directing expression of the gene in a cell that does not naturally express the gene.

[0246] Also provided herein is an isolated nucleotide sequence comprising a gene encoding a PLP-dependent tryptophan decarboxylase from the dicotyledonous plant class.

[0247] Provided herein is an isolated nucleotide sequence comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or a sequence having at least about 70% sequence identity thereto. Optionally, the gene is operably linked to a heterologous promoter capable of directing expression of the gene in a cell that does not naturally express the gene.

[0248] Provided herein is an isolated nucleotide sequence comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least about 70% sequence identity thereto. Optionally, the gene is operably linked to a heterologous promoter capable of directing expression of the gene in a cell that does not naturally express the gene.

[0249] Also provided herein is an isolated nucleotide sequence comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto.

[0250] The present disclosure also provides nucleic acid constructs and vectors comprising heterologous polynucleotide sequences. In particular, nucleic acid constructs and vectors comprising at least one copy (e.g., 1, 2, 3, 4, or more copies) of an exogenous gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure are provided. Such nucleic acid constructs and vectors can be used, for example, to introduce exogenous genes into host cells by any means known to those of skill in the art.

[0251] Thus, a tryptophan decarboxylase gene comprising the nucleic acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or a sequence Provided herein are nucleic acid constructs and vectors comprising a tryptophan decarboxylase gene comprising a nucleic acid sequence set forth in sequence number 23 or 51, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 23 or 51.

[0252] A tryptophan decarboxylase gene comprising the nucleic acid sequence set forth in SEQ ID NO: 8 or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8, or the nucleic acid sequence set forth in SEQ ID NO: 31. Also provided herein are nucleic acid constructs and vectors comprising a tryptophan decarboxylase gene comprising a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:31.

[0253] A tryptophan decarboxylase gene comprising the nucleic acid sequence set forth in SEQ ID NO: 30, or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 30, or the nucleic acid set forth in SEQ ID NO: 47. Also provided herein are nucleic acid constructs and vectors comprising a tryptophan decarboxylase gene comprising a sequence or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:47.

[0254] A tryptophan decarboxylase gene comprising the nucleic acid sequence set forth in SEQ ID NO: 49 or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 49, or the nucleic acid set forth in SEQ ID NO: 51. Also provided herein are nucleic acid constructs and vectors comprising a tryptophan decarboxylase gene comprising a sequence or a sequence having at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:50.

[0255] The tryptophan decarboxylase gene present in the nucleic acid construct or vector can be operably linked to a heterologous promoter, such as those described above herein, and / or other regulatory elements that are operable in the cell into which the vector is to be introduced. The nucleic acid constructs and vectors of the present disclosure can also include at least one copy (e.g., 1, 2, 3, 4 or more copies) of one or more additional genes that are directly or indirectly involved in the biosynthesis of one or more tryptophan derivatives as described above herein.

[0256] In certain embodiments, a vector is provided comprising a gene encoding a PLP-dependent tryptophan decarboxylase of the present disclosure, optionally operably linked to a heterologous promoter capable of directing expression of the gene in a cell that does not naturally express the gene, wherein the tryptophan decarboxylase encoded by the gene is capable of producing at least one tryptophan derivative at a higher level in a cell compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not harbor the gene and contains a PsiD tryptophan decarboxylase. Optionally, the equivalent cell contains a PsiD tryptophan decarboxylase having the amino acid sequence of SEQ ID NO: 9. Optionally, the at least one tryptophan derivative whose production is increased in the cells of the present disclosure is tryptamine or psilocybin. Optionally, the at least one tryptophan derivative whose production is increased in the cells of the present disclosure is N,N-dimethyltryptamine, serotonin, or bufotenin. In one embodiment, the at least one tryptophan derivative whose production is increased in the cells of the present disclosure is 5-methoxy-N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, N-acetylserotonin, and melatonin. In one embodiment, the compounds described herein can also include halogenated, fluorinated, deuterated, and / or substituted variants or fragments thereof.

[0257] Also provided herein are nucleic acid constructs and vectors that include one or more copies of one or more of the above-described additional genes encoding additional tryptophan decarboxylases (e.g., PsiD, AcTDC, AsTDC1, or CrTDC), tryptamine 4-monooxygenases (e.g., PsiH), 4-hydroxytryptamine kinases (e.g., PsiK), methyltransferases (psilocybin synthases) (e.g., PsiM or RmNMT), cytochrome 450 reductases (e.g., Cpr, PcCpr, or OsCPR), tryptamine 5-hydroxylases (e.g., OsT5H), adenosylhomocysteinases (e.g., Sah1), and / or adenosine kinases (e.g., Ado1).

[0258] In the nucleic acid constructs and vectors of the present disclosure, the described genes are typically operably linked to a promoter, optionally a heterologous promoter, and / or other regulatory elements suitable for directing expression of the gene in a host cell, optionally a yeast cell. The promoter can be a constitutive promoter or an inducible promoter. Examples of constitutive promoters useful in yeast cells include, but are not limited to, the PGK (phosphoglycerate kinase) promoter, the ADH-1 (alcohol dehydrogenase) promoter, the ENO (enolase) promoter, the glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter (also referred to as the TDH3 promoter), the constitutive cell wall (CCW) promoter, the histone (HHF) promoter, the hexose transporter (HXT) promoter, the PEP carboxykinase (PCK) promoter, the PYK-1 (pyruvate kinase) promoter, the translation elongation factor-1-alpha (TEF) promoter, and the CYC-1 (cytochrome c oxidase) promoter. In one embodiment, the yeast promoter is a S. cerevisiae promoter. Exemplary constitutive promoters include, but are not limited to, pTDH3, pCCW12, pPGK1, pTEF1, pHHF2, pHXT7, pTEF2, pHHF1, pADH2, pPCK1, pMLS1, pICL1, and pPHO89.

[0259] In another embodiment, the constitutive promoter may not be derived from yeast. Examples of such promoters include, but are not limited to, the cauliflower mosaic virus 35S promoter, the glucocorticoid response element, and the androgen response element. A constitutive promoter may be a naturally occurring molecule or a variant thereof containing, for example, one, two, or three nucleotide substitutions that do not abolish (and preferably enhance) promoter function.

[0260] An exemplary nucleic acid construct or vector described herein comprises psiH, psiK, and psiM, each operably linked to a promoter. Another exemplary nucleic acid construct or vector described herein comprises psiH, psiK, psiM, and Pccpr, each operably linked to a promoter.

[0261] Another exemplary nucleic acid construct or vector described herein includes cnsBv1, psiH, psiK, psiM, and Pccpr, each operably linked to a promoter. Another exemplary nucleic acid construct or vector described herein includes psiD, psiH, psiK, psiM, and Pccpr, each operably linked to a promoter.

[0262] Another exemplary nucleic acid construct or vector described herein comprises SAH1 and ADO1, each operably linked to a promoter. Another exemplary nucleic acid construct or vector described herein comprises SAH1, ADO1, and psiM, each operably linked to a promoter.

[0263] Another exemplary nucleic acid construct or vector described herein comprises RmNMT, OsCPR, and OsT5H, each operably linked to a promoter.

[0264] The nucleic acid constructs and vectors of the present disclosure are typically suitable for transforming or transducing host cells and facilitating expression of one or more genes, either directly or through integration of the genes into the host cell genome. The above nucleic acid constructs and vectors can be utilized in any combination to generate cells according to the present disclosure capable of producing one or more tryptophan derivatives.

[0265] Vectors, such as plasmids, suitable for use in eukaryotic and prokaryotic host cells have been widely described and are well known in the art. Those skilled in the art will understand that vectors may also contain additional sequences and elements useful for replicating the vector in prokaryotic and / or eukaryotic cells, selecting the vector, and expressing heterologous sequences in various host cells. Plasmid vectors, such as pYES-derived vectors, pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors, typically contain additional nucleic acid sequences that allow for easy selection, amplification, and transformation of the expression cassette in cells. Suitable yeast expression vectors include the pPIC series of vectors, yeast integrative plasmids (YIp), yeast replicative plasmids (YRp), yeast centromeric plasmids (YCp), and yeast episomal plasmids (YEp). Additional nucleic acid sequences include an origin of replication that confers autonomous replication of the vector, a selectable marker gene, e.g., encoding antibiotic resistance, a unique multicloning site that provides multiple sites for inserting nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance cell transformation or transduction.

[0266] In some instances, the vector is an expression vector capable of directing gene expression in a cell. Expression vectors are capable of transforming host cells and causing expression of one or more specified polynucleotide molecules. Expression vectors useful for the present disclosure contain regulatory sequences, such as transcriptional control sequences, translational control sequences, origins of replication, and other regulatory sequences, that are compatible with the host cell and control the expression of exogenous genes as described herein. In particular, vectors useful for the present disclosure contain transcriptional control sequences. Transcriptional control sequences are sequences that control the initiation, elongation, and termination of transcription. Particularly important transcriptional control sequences are those that control transcription initiation, such as promoter and enhancer sequences. Suitable transcriptional control sequences include any transcriptional control sequence that can function in a host cell, and the choice of regulatory sequence used can depend on the target host cell. A variety of such transcriptional control sequences are known to those skilled in the art.

[0267] Nucleic acid constructs and vectors can be constructed using known techniques, including, but not limited to, standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. Nucleic acid constructs and vectors can be introduced into host cells using any method known in the art. Thus, the present disclosure also provides host cells containing vectors containing the exogenous genes described herein.

[0268] To facilitate identification of transformants when a nucleic acid construct or vector as described herein is introduced into a cell, the vector desirably contains a selectable or screenable marker gene in addition to the exogenous gene. The marker gene confers a unique phenotype on cells expressing the marker gene, thus allowing such transformed cells to be distinguished from cells that do not possess the marker. A selectable marker gene confers a trait for which one can "select" based on resistance to a selective agent (e.g., an antibiotic or other treatment that is damaging to untransformed cells). A screenable marker gene (or reporter gene) confers a trait that can be identified through observation or testing, i.e., by "screening" (e.g., β-glucuronidase, luciferase, green fluorescent protein (GFP), or other enzymatic activity not present in untransformed cells). The marker gene and the nucleotide sequence of interest do not need to be linked. The actual choice of marker is not important, as long as it is functional (i.e., selectable) in combination with the selected cells. Examples of selectable markers are markers that confer antibiotic resistance, such as hygromycin, nourseothricin, ampicillin, erythromycin, chloramphenicol or tetracycline resistance.

[0269] In one example, marker-free integration method is used for genome integration.For example, marker-free integration method can include CRISPR / Cas system.The method for marker-free integration using CRISPR / Cas system is known in the art (such as those described in Chi et al., 2019 and Jessop-Fabre et al., 2016: Biotechnol J).

[0270] Any method can be used to introduce a nucleic acid construct or vector containing an exogenous gene into a cell, and many such methods are well known to those of skill in the art. Yeast cells are typically transformed by chemical methods. For example, cells are transformed by about 10 4 They can be treated with lithium acetate to achieve a transformation efficiency of colony-forming units (transformed cells) / μg of DNA. Other common procedures for transforming yeast include utilizing the generation of yeast spheroplasts, the biolistic method in which DNA-coated metal microprojectiles are bombarded into cells, and the glass bead method, which relies on agitation of yeast cells with glass beads and the DNA to be delivered to the cells. Of course, any suitable means of introducing nucleic acids into cells, such as yeast cells, can be used.

[0271] Recombinant DNA technology can be used to improve the expression of transformed nucleic acid molecules, including exogenous genes, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resulting transcripts are translated, and the efficiency of post-translational modifications, for example, by manipulating the copy number of the nucleic acid molecule in the host cell. Recombinant techniques useful for increasing expression of the nucleic acid molecules defined herein include, but are not limited to, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of stability sequences to mRNA, substitution or modification of transcriptional control signals (e.g., promoters, operators, enhancers), substitution or modification of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of the polynucleotide molecule to accommodate the codon usage of the host cell, and deletion of sequences that destabilize the transcript.

[0272] The nucleic acid construct or vector containing the exogenous gene can be contained in the cell in any form. For example, the nucleic acid construct can be integrated into the genome of the cell (e.g., by homologous recombination or random integration) or maintained in an episomal state that can be stably transmitted to daughter cells. Such extrachromosomal genetic elements (such as plasmids) can additionally contain a selectable marker that ensures the presence of such genetic elements in daughter cells. Furthermore, the cell can be stably or transiently transformed. Additionally, the cell can contain a single copy or multiple copies of the nucleic acid molecule.

[0273] Unless otherwise indicated, the recombinant DNA and cell culture techniques described and utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991), D.M.G.lover and B.D. Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996), and F.M.A.usubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988).

[0274] In order that the present disclosure may be readily understood and put into practice, certain preferred embodiments will now be described by way of the following non-limiting examples.

[0275] Reference herein to any prior publication (or information derived therefrom) or to any matter that is publicly known is not, and should not be taken as, an acknowledgment or admission, or any form of suggestion, that the prior publication (or information derived therefrom) or publicly known matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

[0276] The disclosure is further described in the following numbered paragraphs:

[0277] 1. A cell comprising an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a fungal pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase derived from the class Eurotium, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to the fungal PLP-dependent tryptophan decarboxylase or contains one or more conservative amino acid substitutions thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not possess the exogenous gene and contains a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase derived from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical to the fungal PLP-dependent tryptophan decarboxylase or contains one or more conservative amino acid substitutions thereto.

[0278] 2. A cell containing an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or a sequence having at least about 70% sequence identity thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not possess the exogenous gene and contains a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase derived from the class Eurotium or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0279] 3. The cell of paragraph 2, wherein the tryptophan decarboxylase encoded by the exogenous gene comprises the amino acid sequence set forth in SEQ ID NO:1.

[0280] 4. A cell comprising an exogenous gene and capable of producing at least one tryptophan derivative, wherein the exogenous gene encodes a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO: 7 or a sequence having at least about 70% sequence identity thereto, and the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in an equivalent cell that does not possess the exogenous gene and comprises a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase derived from the class Eurotium or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0281] 5. The cell of any one of paragraphs 1-4, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of the at least one tryptophan derivative in a comparable cell that does not harbor the exogenous gene and that contains a non-PLP-dependent tryptophan decarboxylase.

[0282] 6. The cell of any one of paragraphs 1-5, wherein the non-PLP-dependent tryptophan decarboxylase is a PsiD tryptophan decarboxylase, optionally comprising the amino acid sequence set forth in SEQ ID NO:9.

[0283] 7. The cell of any one of paragraphs 1 through 6, wherein the cell contains two or more copies of the exogenous gene.

[0284] 8. The cell of any one of paragraphs 1-7, wherein the exogenous gene is operably linked to a heterologous promoter.

[0285] 9. The cell of any one of paragraphs 1-8, further comprising a nucleic acid sequence encoding an additional tryptophan decarboxylase.

[0286] 10. The cell of paragraph 9, wherein the additional tryptophan decarboxylase is optionally PsiD comprising the amino acid sequence set forth in SEQ ID NO: 9, or a sequence having at least about 70% sequence identity thereto.

[0287] 11. The cell of any one of paragraphs 1 to 10, wherein the cell further comprises a nucleic acid sequence encoding a tryptamine 4-monooxygenase comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least about 70% sequence identity thereto.

[0288] 12. The cell of paragraph 11, wherein the tryptamine 4-monooxygenase is PsiH.

[0289] 13. The cell of paragraph 12, comprising two or more copies of a gene encoding PsiH.

[0290] 14. The cell of any one of paragraphs 1 to 13, wherein the cell further comprises a nucleic acid sequence encoding a 4-hydroxytryptamine kinase comprising the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least about 70% sequence identity thereto.

[0291] 15. The cell of paragraph 14, wherein the 4-hydroxytryptamine kinase is PsiK.

[0292] 16. The cell of paragraph 15, comprising two or more copies of a gene encoding PsiK.

[0293] 17. The cell of any one of paragraphs 1-16, wherein the cell further comprises a nucleic acid sequence encoding a methyltransferase comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least about 70% sequence identity thereto.

[0294] 18. The cell of paragraph 17, wherein the methyltransferase is PsiM.

[0295] 19. The cell of paragraph 18, comprising two or more copies of a gene encoding PsiM.

[0296] 20. The cell of any one of paragraphs 1 to 19, wherein the cell further comprises a nucleic acid sequence encoding a cytochrome P450 reductase comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least about 70% sequence identity thereto.

[0297] 21. The cell of paragraph 20, wherein the cytochrome P450 reductase is PcCpr.

[0298] 22. The cell of paragraph 21, comprising two or more copies of a gene encoding PcCpr.

[0299] 23. The cell of any one of paragraphs 1 to 22, wherein the cell further comprises one or more copies, optionally at least two copies, of a gene encoding an adenosylhomocysteinase comprising the amino acid sequence set forth in SEQ ID NO: 19, or a sequence having at least about 70% sequence identity thereto.

[0300] 24. The cell of any one of paragraphs 1 to 23, wherein the cell further comprises one or more copies, optionally at least two copies, of a gene encoding an adenosine kinase comprising the amino acid sequence set forth in SEQ ID NO: 21, or a sequence having at least about 70% sequence identity thereto.

[0301] 25. The cell of any one of paragraphs 1 to 24, wherein the cell is a yeast cell.

[0302] 26. The cell of paragraph 25, wherein the yeast cell is a cell of a Saccharomyces species.

[0303] 27. The cell of paragraph 26, wherein the species of Saccharomyces is S. cerevisiae.

[0304] 28. The cell of any one of paragraphs 1 to 27, wherein the at least one tryptophan derivative is tryptamine or a tryptamine-derived alkaloid.

[0305] 29. The cell of paragraph 28, wherein the tryptophan derivative is tryptamine.

[0306] 30. The cell of paragraph 28, wherein the tryptamine-derived alkaloid is psilocybin.

[0307] 31. (a) one or more copies of a gene encoding a fungal PLP-dependent tryptophan decarboxylase from the class Eurotium, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to, or contains one or more conservative amino acid substitutions relative to, said fungal PLP-dependent tryptophan decarboxylase; (b) one or more copies of a gene encoding PsiH tryptamine 4-monooxygenase; (c) one or more copies of a gene encoding the PsiK hydroxytryptamine kinase; and (d) one or more copies of a gene encoding a PsiM methyltransferase A cell capable of producing at least one tryptophan derivative, comprising:

[0308] 32. (a) one or more copies of a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a sequence having at least about 70% sequence identity thereto; (b) one or more copies of a gene encoding PsiH tryptamine 4-monooxygenase; (c) one or more copies of the gene encoding the PsiK hydroxytryptamine kinase; (d) one or more copies of a gene encoding a PsiM methyltransferase A cell capable of producing at least one tryptophan derivative, comprising:

[0309] 33. (a) one or more copies of a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto; (b) one or more copies of a gene encoding PsiH tryptamine 4-monooxygenase; (c) one or more copies of the gene encoding the PsiK hydroxytryptamine kinase; (d) one or more copies of a gene encoding a PsiM methyltransferase A cell capable of producing at least one tryptophan derivative, comprising:

[0310] 34. The cell of any one of paragraphs 31-33, wherein the cell is capable of a higher level of production of at least one tryptophan derivative compared to a production level of the at least one tryptophan derivative in a comparable cell that does not possess (a) and that includes a tryptophan decarboxylase encoded by a gene other than (a), optionally a gene encoding a PsiD tryptophan decarboxylase.

[0311] 35. The cell of any one of paragraphs 31 to 34, wherein the tryptophan derivative is tryptamine or psilocybin.

[0312] 36. The cell of any one of paragraphs 31 to 35, wherein the cell further comprises one or more copies of a gene encoding a cytochrome P450 reductase, optionally a PcCpr cytochrome P450 reductase.

[0313] 37. The cell of any one of paragraphs 31 to 36, wherein the cell further comprises one or more copies of a gene encoding an adenosylhomocysteinase, optionally a Sah1 adenosylhomocysteinase.

[0314] 38. The cell of any one of paragraphs 31 to 37, wherein the cell further comprises one or more copies of a gene encoding an adenosine kinase, optionally Ado1 adenosine kinase.

[0315] 39. The cell of any one of paragraphs 31 to 38, wherein the cell further comprises one or more copies of a gene encoding an additional tryptophan decarboxylase, optionally a PsiD and / or a CrTDC tryptophan decarboxylase.

[0316] 40. A vector comprising a gene encoding a fungal PLP-dependent tryptophan decarboxylase from the class Eurotium, or a PLP-dependent tryptophan decarboxylase that is at least about 80% identical to, or contains one or more conservative amino acid substitutions thereto, optionally operably linked to a heterologous promoter capable of directing expression of the gene in a cell that does not naturally express the gene, wherein the tryptophan decarboxylase encoded by the gene is capable of producing a higher level of at least one tryptophan derivative in a cell compared to the production level of at least one tryptophan derivative in an equivalent cell that does not possess the gene and contains a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical to, or contains one or more conservative amino acid substitutions thereto.

[0317] 41. A vector comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a sequence having at least about 70% sequence identity thereto, optionally operably linked to a heterologous promoter capable of directing expression of said gene in a cell that does not naturally express said gene, wherein the tryptophan decarboxylase encoded by said gene is capable of a higher level of production of at least one tryptophan derivative in a cell compared to a production level of at least one tryptophan derivative in an equivalent cell that does not possess said gene and that contains a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium, or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0318] 42. A vector comprising a gene encoding a tryptophan decarboxylase having the amino acid sequence set forth in SEQ ID NO:7, or a sequence having at least about 70% sequence identity thereto, optionally operably linked to a heterologous promoter capable of directing expression of said gene in a cell that does not naturally express said gene, wherein the tryptophan decarboxylase encoded by said gene is capable of a higher level of production of at least one tryptophan derivative in a cell compared to a production level of at least one tryptophan derivative in an equivalent cell that does not possess said gene and that contains a tryptophan decarboxylase other than a PLP-dependent tryptophan decarboxylase from the class Eurotium or a tryptophan decarboxylase that is at least about 80% identical thereto or contains one or more conservative amino acid substitutions thereto.

[0319] 43. A host cell containing the vector of any one of paragraphs 40 to 42.

[0320] 44. A method for producing at least one tryptophan derivative, comprising culturing the cell of any one of paragraphs 1-39 or 43 under conditions suitable for the production of at least one tryptophan derivative.

[0321] 45. The method of paragraph 44, further comprising the step of extracting at least one tryptophan derivative from the cells.

[0322] 46. ​​The method of paragraph 45, wherein the extracted tryptophan derivative is tryptamine or a tryptamine-derived alkaloid.

[0323] 47. The method of paragraph 46, wherein the tryptophan derivative is tryptamine.

[0324] 48. The method of paragraph 46, wherein the tryptamine-derived alkaloid is psilocybin.

[0325] 49. A tryptophan derivative produced by the method of any one of paragraphs 44 to 48.

[0326] 50. A composition comprising the cells of any one of paragraphs 1 to 39 or 43 or the extracted tryptophan derivative of paragraph 49.

[0327] 51. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject the composition of paragraph 50.

[0328] 52. The method of paragraph 51, wherein the disease or disorder is a neurodevelopmental, neurological, neuromuscular, movement, psychiatric, or psychological disease, disorder, or syndrome. [Example]

[0329] [Example 1] Plasmid assembly and strain construction The tryptophan decarboxylase genes cnsBv1 (SEQ ID NO: 2) and cnsBv2 (SEQ ID NO: 4) from Penicillium expansum (Lin et al., 2015, Angew Chem Int Ed Engl, 54:3004-7), the tryptophan decarboxylase gene represented by SEQ ID NO: 8 from Aspergillus steinii (referred to herein as "asTDC1"), psiD, psiH, psiK, and psiM from Aspergillus stainii FSU12409 (Fricke et al., 2017, Angew Chem Int Ed Engl, 56:12352-55; GenBank:KY984101.1, GenBank:MF000993.1, GenBank:KY984099.1, GenBank:KY984100.1, respectively), and Pccpr (Milne et al., Metab Eng. 60:25-36) were synthesized as synthetic gene fragments by Twist Biosciences. The wild-type cnsBv1, cnsBv2, ΔcnsBv2, asTDC1, psiD, psiH, psiK, psiM, and Pccpr coding sequences are provided in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18, respectively. The amino acid sequences of the CnsBv1, CnsBv2, ΔCnsBv2, AsTDC1, PsiD, PsiH, PsiK, PsiM, and Pccpr polypeptides encoded by these coding sequences are provided in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17, respectively.

[0330] The coding sequence of the S-adenosylhomocysteine ​​hydrolyse SAH1 gene from S. cerevisiae (GenBank: NM_001178934.3) was amplified by PCR from S. cerevisiae genomic DNA using primers 5'-GGTCTCTAGGATGTCTGCTCCAGCTCAAAACT-3' (SAH1F; SEQ ID NO: 32) and 5'-GGTCTCAAACTTCAATATCTGTAGTGGTCGGCC-3' (SAH1R; SEQ ID NO: 33). The coding sequence of the adenosine kinase ADO1 gene from S. cerevisiae (GenBank NP_010961.3) was amplified by PCR from S. cerevisiae genomic DNA using primers 5'-GGTCTCTGCAATGACCGCACCATTGGTAG-3' (ADO1F; SEQ ID NO: 34) and 5'-GGTCTCATGTGCTATTTAGAGTAAGATATTTTTTCGGAAGG-3' (ADO1R; SEQ ID NO: 35). The wild-type SAH1 and ADO1 coding sequences are provided in SEQ ID NO: 20 and SEQ ID NO: 22, respectively. The amino acid sequences of Sah1 and Ado1 are provided in SEQ ID NO: 19 and SEQ ID NO: 21, respectively.

[0331] An alignment of the CnsB and PsiD polypeptide sequences is shown in Figure 3. Based on pairwise alignment using the Needleman-Wunsch algorithm, CnsBv1 and PsiD share only 16.0% sequence identity, CnsBv2 and PsiD share only 16.2% sequence identity, and ΔCnsBv2 and PsiD share only 15.3% sequence identity. An alignment of the CnsB amino acid sequence (SEQ ID NOS: 1, 3, and 5) with that of CrTDC (SEQ ID NOS: 29), a tryptophan decarboxylase from Catharanthus roseus, is shown in Figure 4. The sequence identity is 24.3% between CnsBv1 and CrTDC, 23.5% between CnsBv2 and CrTDC, and 23.5% between ΔCnsBv2 and CrTDC. PsiD and CrTDC share 15.9% sequence identity.

[0332] The cnsBv1, asTDC1, psiD, psiH, psiK, psiM, and Pccpr coding sequences were codon-optimized for expression in Saccharomyces cerevisiae and equipped with 5' and 3' restriction sites for Golden Gate cloning. The codon-optimized coding sequences for cnsBv1 and asTDC1 are provided in SEQ ID NOs: 23 and 31, respectively. The codon-optimized coding sequences for psiD, psiH, psiK, psiM, and Pccpr are provided in SEQ ID NOs: 24-28, respectively.

[0333] Plasmids pNMT-cnsBv1, pNMT-asTDC1, and pNMT-psiD were constructed from individual parts using BsaI Golden Gate assembly (Engler et al., 2008, PLoS One, 3:e3647). The plasmids are identical except for the tryptophan decarboxylase gene and contain the following components: the pTDH3 promoter and tENO1 terminator flanking each decarboxylase gene (operably linked to the pTDH3 promoter), an ampicillin resistance gene, a ColE1 origin of replication, a URA3 auxotrophic marker, and homology arms for integration of the expression cassette into the URA3 locus of the S. cerevisiae genome. E. coli 10-β cells were used for plasmid cloning and propagation (grown at 37°C and 180 rpm).

[0334] Plasmids pNMT-BHKMC and pNMT-DHKMC were constructed from individual parts using BsaI Golden Gate assembly. The plasmids are identical except for the tryptophan decarboxylase gene and contain the following components: the pTDH3 promoter and tENO1 terminator flanking each decarboxylase gene (pNMT-BHKMC contains cnsBv1 operably linked to the pTDH3 promoter; pNMT-DHKMC contains psiD operably linked to the pTDH3 promoter); the pCCW12 promoter and tSSA1 terminator flanking psiH (operably linked to the pCCW12 promoter); Pccpr (pPGK1 promoter) the pPGK1 promoter and tADH1 terminator flanked by psiK (operably linked to the pHHF2 promoter); the pTEF1 promoter and tENO2 terminator flanked by psiM (operably linked to the pTEF1 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a URA3 auxotrophic marker; and homology arms for integration of the expression cassette into the URA3 locus of the S. cerevisiae genome.

[0335] Plasmid pNMT-HKMC was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pCCW12 promoter and tSSA1 terminator flanked by psiH (operably linked to the pCCW12 promoter); the pPGK1 promoter and tADH1 terminator flanked by Pccpr (operably linked to the pPGK1 promoter); the pHHF2 promoter and tPGK1 terminator flanked by psiK (operably linked to the pHHF2 promoter); the pTEF1 promoter and tENO2 terminator flanked by psiM (operably linked to the pTEF1 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a LEU2 auxotrophic marker; and homology arms for expression cassette integration into the LEU2 locus of the S. cerevisiae genome.

[0336] Plasmid pNMT-MASB was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pTDH3 promoter and tENO1 terminator flanked by cnsBv1 (operably linked to the pTDH3 promoter); the pTEF1 promoter and tENO2 terminator flanked by psiM (operably linked to the pTEF1 promoter); the pCCW12 promoter and tSSA1 terminator flanked by ADO1 (operably linked to the pCCW12 promoter); the pTDH3 promoter and tENO1 terminator flanked by SAH1 (operably linked to the pTDH3 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a HIS3 auxotrophic marker; and homology arms for expression cassette integration into the HIS3 locus of the S. cerevisiae genome.

[0337] Plasmid pNMT-MAS was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pTEF1 promoter and tENO2 terminator flanked by psiM (operably linked to the pTEF1 promoter); the pCCW12 promoter and tSSA1 terminator flanked by ADO1 (operably linked to the pCCW12 promoter); the pTDH3 promoter and tENO1 terminator flanked by SAH1 (operably linked to the pTDH3 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a HIS3 auxotrophic marker; and homology arms for expression cassette integration into the HIS3 locus of the S. cerevisiae genome.

[0338] Plasmid pNMT-M was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pTEF1 promoter and tENO2 terminator flanked by psiM (operably linked to the pTEF1 promoter), an ampicillin resistance gene, a ColE1 origin of replication, a HIS3 auxotrophic marker, and homology arms for expression cassette integration into the HIS3 locus of the S. cerevisiae genome.

[0339] S. cerevisiae strain BY4741 (grown at 30°C and 250 rpm) was used as the parent strain for all transformations. All transformations of S. cerevisiae were performed using the LiAc / SS-carried DNA / PEG method (Gietz and Schiestl, 2007, Nature Protocols, 2:1-4).

[0340] Transformation of S. cerevisiae with pNMT-cnsBv1, pNMT-asTDC1, and pNMT-psiD yielded strains SC-cnsBv1, SC-asTDC1, and SC-psiD, respectively, each containing a single copy of the relevant tryptophan decarboxylase gene.

[0341] Transformation of S. cerevisiae with both pNMT-DHKMC and pNMT-HKMC yielded strain SC-D, and transformation with both pNMT-BHKMC and pNMT-HKMC yielded strain SC-B. Strain SC-D contains a single copy of psiD and two copies of each of psiH, psiK, psiM, and Pccpr. Strain SC-B contains a single copy of cnsBv1 and two copies of each of psiH, psiK, psiM, and Pccpr.

[0342] Transformation of S. cerevisiae with pNMT-DHKMC, pNMT-HKMC, and pNMT-M together yielded strain SC-DM, and transformation with pNMT-BHKMC, pNMT-HKMC, and pNMT-M together yielded strain SC-BM. Strain SC-DM contains a single copy of psiD, two copies each of psiH, psiK, and Pccpr, and three copies of psiM. Strain SC-BM contains a single copy of cnsB, two copies each of psiH, psiK, and Pccpr, and three copies of psiM.

[0343] Transformation of S. cerevisiae with pNMT-DHKMC, pNMT-HKMC, and pNMT-MAS together yielded strain SC-DMAS, while transformation with pNMT-BHKMC, pNMT-HKMC, and pNMT-MAS together yielded strain SC-BMAS. Strain SC-DMAS contains a single copy of psiD, two copies each of psiH, psiK, and Pccpr, three copies of psiM, and a second copy each of the endogenous S. cerevisiae genes SAH1 and ADO1. Strain SC-BMAS contains a single copy of cnsB, two copies each of psiH, psiK, and Pccpr, three copies of psiM, and a second copy each of the endogenous S. cerevisiae genes SAH1 and ADO1.

[0344] Transformation of S. cerevisiae with pNMT-DHKMC, pNMT-HKMC, and pNMT-MASB together yielded strain SC-DMASB, while transformation with pNMT-BHKMC, pNMT-HKMC, and pNMT-MASB together yielded strain SC-BMASB. Strain SC-DMASB contains a single copy of psiD, a single copy of cnsB, two copies each of psiH, psiK, and Pccpr, three copies of psiM, and a second copy each of the endogenous S. cerevisiae genes SAH1 and ADO1. Strain SC-BMASB contains two copies of cnsB, two copies each of psiH, psiK, and Pccpr, three copies of psiM, and a second copy each of the endogenous S. cerevisiae genes SAH1 and ADO1.

[0345] [Example 2] Tryptamine biosynthesis The ability of S. cerevisiae strains SC-cnsBv1 and SC-psiD to produce tryptamine was determined. Two-mL cultures of dropout medium (6.7 g / L amino acid-free yeast nitrogen base, 1.4 g / L trp / leu / his / ura-free yeast synthetic dropout medium, 20 g / L glucose, 76 mg / L tryptophan, 500 mg / L leucine, 125 mg / L histidine) were inoculated with a single colony of S. cerevisiae. After overnight growth, 2 mL of 2x YPD (40 g / L glucose, 40 g / L peptone, 2 g / L yeast extract) was added to each culture.

[0346] Samples of 400 μL of culture medium were taken on days 5 and 6, and cell and medium extractions were performed separately after separation by centrifugation. The cell pellet was extracted by adding 400 μL of methanol, followed by thorough vortexing, centrifugation, transfer of the supernatant to a new microcentrifuge tube, drying, and resuspension in 200 μL of methanol (cell extract). The culture medium was treated with 500 μL of n-butanol, and the organic layer was transferred to a new microcentrifuge tube, dried, and resuspended in 200 μL of methanol (cell growth medium extract).

[0347] For analysis, 4 μL of cell extract or 3 μL of cell growth medium extract was injected into an Agilent 1260 liquid chromatography system equipped with a Kinetex C18 column (2.1 × 100 mm, 2.6 μm) coupled to an Agilent 6130 quadrupole mass spectrometer with an ESI source. The mobile phase was a 10-minute linear gradient of 5 to 95% acetonitrile-water containing 0.1% formic acid. Tryptamine peaks in the extract LC-MS chromatograms were identified by comparison with the chromatogram of an authenticated tryptamine standard. To determine tryptamine titer, known amounts of tryptamine were injected into the LC-MS, and the corresponding peak areas were used to generate a standard curve. The standard curve was then used to calculate tryptamine titer in the extract from the observed tryptamine chromatogram peak areas.

[0348] As shown in Table 2 and Figure 5, tryptamine production was significantly higher at both time points (days 5 and 6) using the SC-cnsBv1 strain compared to the SC-psiD strain, as determined in both cells and growth medium.

[0349] In separate experiments, the ability of S. cerevisiae strains SC-cnsBv1, SC-asTDC1, and SC-psiD to produce tryptamine was determined. A 1 mL culture of YPD (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) was inoculated with a single colony of S. cerevisiae. 100 μL samples of the culture medium were taken on days 3, 5, and 6. Extraction was performed by adding 200 μL of methanol followed by thorough vortexing. The extract was centrifuged extensively to remove cellular debris.

[0350] For analysis, 1 μL of the extract was injected into an Agilent 1260 liquid chromatography system equipped with a Kinetex C18 column (2.1 × 100 mm, 2.6 μm) coupled to an Agilent 6130 quadrupole mass spectrometer with an ESI source. The mobile phase was a 10-minute linear gradient of 5 to 95% acetonitrile-water containing 0.1% formic acid. Tryptamine peaks in the extract LC-MS chromatogram were identified by comparison with the chromatogram of an authenticated tryptamine standard. To determine tryptamine titer, known amounts of tryptamine were injected into the LC-MS, and the corresponding peak areas were used to generate a standard curve. The standard curve was then used to calculate tryptamine titer in the extract from the observed tryptamine chromatogram peak areas.

[0351] As shown in Figure 7, tryptamine production in the presence of asTDC1 was significantly higher than in the presence of cnsBv1 or psiD at all three time points (days 3, 5, and 6).

[0352] Example 3: Biosynthesis of psilocybin and psilocin The ability of S. cerevisiae strains SC-D, SC-B, SC-DM, SC-BM, SC-DMAS, SC-BMAS, SC-BMASB, and SC-DMASB to produce psilocybin and psilocin was determined. 4 mL cultures of dropout medium (adjusted to pH 4 and containing 20 g / L KH2PO4 and 1 μM thiamine) were inoculated with a single colony of S. cerevisiae. 400 μL culture samples were taken on days 2 and 3, and cell and medium extractions were performed separately after separation by centrifugation. The cell pellet was extracted by adding 400 μL of methanol, followed by thorough vortexing, centrifugation, transfer of the supernatant to a new microcentrifuge tube, drying, and resuspension in 200 μL of methanol. The culture medium was washed twice with hexane, dried, and resuspended in 200 μL of methanol.

[0353] For analysis, 1 μL of cell extract or 1 μL of medium extract was injected onto a Thermo Scientific Vanquish ultra-high performance liquid chromatography system equipped with an Agilent C18 column (2.1 × 100 mm, 2.7 μm) coupled to an Orbitrap Exploris 120 mass spectrometer. The mobile phase was a 10-min linear gradient of 5–95% acetonitrile-water containing 0.1% formic acid.

[0354] As shown in Table 3 and Figure 8, psilocybin and psilocin production was slightly higher as determined in growth medium using strain SC-B compared to strain SC-D. The difference in psilocybin and psilocin production did not correspond to the significantly higher tryptamine production shown in Table 2 and Figure 6, suggesting a bottleneck in the psilocybin pathway downstream of tryptophan decarboxylase.

[0355] As shown in Table 4 and Figure 9, the addition of a third copy of psiM in SC-BM and SC-DM resulted in a greater difference in psilocybin and psilocin production, as determined in both cells and growth medium, using the SC-BM strain compared to the SC-DM strain.

[0356] As shown in Table 5 and Figure 10, the addition of a second copy of each of the native S. cerevisiae genes SAH1 and ADO1 in SC-BMAS and SC-DMAS resulted in an even greater difference in psilocybin and psilocin production using the SC-BM strain compared to the SC-DM strain, as well as a significant increase in overall production using the SC-DMAS strain compared to the SC-DM strain and the SC-BMAS strain compared to the SC-BM strain, as shown in Figure 12. This indicated that the bottleneck in tryptamine conversion to psilocybin present in strains SC-B and SC-BM was alleviated in strain SC-BMAS. Therefore, the significant difference in psilocybin and psilocin production using the SC-BMAS strain compared to the SC-DMAS strain was the result of the higher tryptamine production rate of cnsBv1 compared to psiD.

[0357] As shown in Table 6, Figure 11, and Figure 12, the addition of a copy of cnsBv1 in SC-BMASB and SC-DMASB resulted in a large increase in production using the SC-DMAS strain compared to the SC-DMASB strain, and a slight increase using the SC-BMAS strain compared to the SC-BMASB strain, indicating that a single copy of cnsBv1 produced enough tryptamine to nearly saturate the downstream pathway enzymes psiH, psiK, and psiM, while psiD only supplied a small amount of tryptamine that the downstream enzymes could convert.

[0358] [Example 4] N-methyltransferase Plasmid assembly and strain construction The coding sequences for osT5H, osCPR, and rmNMT (Chen X, Li J, Yu L, et al., Bioproduction platform using a novel cane toad (Rhinella marina) N-methyltransferase for psychedelic-inspired drug discovery. Research Square; 2023. DOI: 10.21203 / rs.3.rs-2667175 / v1.) were synthesized by Twist Biosciences as synthetic gene fragments.

[0359] The wild-type osT5H, osCPR, and rmNMT coding sequences are provided in SEQ ID NOs: 37, 40, and 43, respectively. The amino acid sequences of the OsT5H, OsCPR, and RmNMT polypeptides encoded by these coding sequences are provided in SEQ ID NOs: 36, 39, and 42, respectively.

[0360] The osT5H, osCPR, and rmNMT coding sequences were codon-optimized for expression in Saccharomyces cerevisiae and provided with 5' and 3' restriction sites for Golden Gate cloning. The codon-optimized coding sequences for osT5H, osCPR, and rmNMT are provided in SEQ ID NOs: 38, 41, and 44, respectively.

[0361] Plasmid pNMT-rmNMT1 was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pTDH3 promoter and tTDH1 terminator flanked by asTDC1 (operably linked to the pTDH3 promoter); the pPGK1 promoter and tSSA1 terminator flanked by osCPR (operably linked to the pPGK1 promoter); the pCCW12 promoter and tPGK1 terminator flanked by osT5H (operably linked to the pCCW12 promoter); the pTEF2 promoter and tENO1 terminator flanked by rmNMT (operably linked to the pTEF2 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a URA3 auxotrophic marker; and homology arms for expression cassette integration into the URA3 locus of the S. cerevisiae genome.

[0362] Plasmid pNMT-RmNMT2 was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pTDH3 promoter and tTDH1 terminator flanked by asTDC1 (operably linked to the pTDH3 promoter); the pTEF2 promoter and tENO1 terminator flanked by rmNMT (operably linked to the pTEF2 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a URA3 auxotrophic marker; and homology arms for expression cassette integration into the URA3 locus of the S. cerevisiae genome.

[0363] Plasmid pNMT-rmNMT3 was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pTDH3 promoter and tTDH1 terminator flanked by cnsBv1 (operably linked to the pTDH3 promoter); the pPGK1 promoter and tSSA1 terminator flanked by osCPR (operably linked to the pPGK1 promoter); the pCCW12 promoter and tPGK1 terminator flanked by osT5H (operably linked to the pCCW12 promoter); the pTEF2 promoter and tENO1 terminator flanked by rmNMT (operably linked to the pTEF2 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a URA3 auxotrophic marker; and homology arms for expression cassette integration into the URA3 locus of the S. cerevisiae genome.

[0364] Plasmid pNMT-RmNMT4 was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pTDH3 promoter and tTDH1 terminator flanked by cnsBv1 (operably linked to the pTDH3 promoter); the pTEF2 promoter and tENO1 terminator flanked by rmNMT (operably linked to the pTEF2 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a URA3 auxotrophic marker; and homology arms for expression cassette integration into the URA3 locus of the S. cerevisiae genome.

[0365] Plasmid pNMT-RmNMT5 was constructed from individual parts using BsaI Golden Gate assembly and contains the following components: the pPGK1 promoter and tSSA1 terminator flanked by osCPR (operably linked to the pPGK1 promoter); the pCCW12 promoter and tPGK1 terminator flanked by osT5H (operably linked to the pCCW12 promoter); the pTEF2 promoter and tENO1 terminator flanked by rmNMT (operably linked to the pTEF2 promoter); an ampicillin resistance gene; a ColE1 origin of replication; a URA3 auxotrophic marker; and homology arms for expression cassette integration into the URA3 locus of the S. cerevisiae genome.

[0366] Transformation of S. cerevisiae with pNMT-rmNMT1-5 resulted in strains SC-rmNMT1-5, respectively. S. cerevisiae strain BY4741 (grown at 30°C and 250 rpm) was used as the parent strain for all transformations. All transformations of S. cerevisiae were performed using the LiAc / SS carrier DNA / PEG method (Gietz and Schiestl, 2007, Nature Protocols, 2:1-4).

[0367] Biosynthesis The ability of S. cerevisiae strains SC-rmNMT1 1-5 to produce tryptamine, 5-OH-tryptophan, serotonin, N,N-dimethyltryptamine, and bufotenin was determined. A 1 mL culture of YPD (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) was inoculated with a single colony of S. cerevisiae. A 100 μL culture sample was taken on day 6. Extraction was performed by adding 200 μL of methanol followed by thorough vortexing. The extract was thoroughly centrifuged to remove cellular debris.

[0368] For analysis, 1 μL of the extract was injected onto a Vanquish UPLC system equipped with a Kinetex C18 column (2.1 × 100 mm, 2.6 μm) coupled to an Orbitrap Exploris 120 MS (OE120) mass spectrometer with an ESI source. The mobile phase was a 10-minute linear gradient of 5 to 95% acetonitrile-water containing 0.1% formic acid. Tryptamine, 5-OH-tryptophan, serotonin, and N,N-dimethyltryptamine peaks in the extracted LC-MS chromatogram were identified by comparison with the extracted chromatograms of tryptamine, 5-OH-tryptophan, serotonin, and N,N-dimethyltryptamine certified standards.

[0369] As shown in Figure 14, PLP-dependent tryptophan carboxylases such as cnsBv1 and asTDC1 can be used to provide tryptamine for the production of serotonin, N,N-dimethyltryptamine, and bufotenin in the context of the appropriate biosynthetic pathways.

[0370] [Example 5] E. coli containing asTDC1 Plasmid assembly and strain construction The coding sequences for psiD and asTDC1 were synthesized by Twist Biosciences as synthetic gene fragments.

[0371] The psiD and asTDC1 coding sequences were codon-optimized for expression in E. coli and inserted into the NdeI-XhoI restriction sites of pET28a. The codon-optimized coding sequences of psiD and asTDC1 are provided in SEQ ID NOs: 45 and 46, respectively.

[0372] Transformation of E. coli BL21(DE3) with pET28a-psiD, pET28a-asTDC1, and pET28a resulted in strains EC-psiD, EC-asTDC1, and EC-ctrl, respectively.

[0373] The ability of E. coli to produce tryptamine, psilocybin, and psilocin is determined essentially as described in Examples 2 and 4.

[0374] Tryptamine biosynthesis The ability of E. coli strains EC-psiD, EC-asTDC1, and EC-ctrl to produce tryptamine was determined. A 1 mL culture of lysogeny broth (LB) (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract) was inoculated with a single colony of E. coli and incubated at 37°C for 6 hours. 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added. A 100 μL culture sample was taken on day 3. Extraction was performed by adding 200 μL of methanol followed by thorough vortexing. The extract was thoroughly centrifuged to remove cellular debris.

[0375] LC-MS-based analysis and quantification of tryptamine yield were performed as described in Examples 2 and 4.

[0376] As shown in Figure 15, a representative PLP-dependent tryptophan carboxylase, asTDC1, is active in E. coli and produces approximately 50% more tryptamine than PsiD.

[0377] Example 6: S. cerevisiae containing crTDC, AcTDC, and cnsBv2 Plasmid assembly and strain construction The coding sequences for crTDC, acTDC and cnsBv2 were synthesized by Twist Biosciences as synthetic gene fragments.

[0378] The wild-type crTDC and acTDC coding sequences are provided in SEQ ID NOs: 30 and 50, respectively. The amino acid sequences of the CrTDC and AcTDC polypeptides encoded by these coding sequences are provided in SEQ ID NOs: 29 and 48, respectively.

[0379] The crTDC, acTDC, and cnsBv2 coding sequences were codon-optimized for expression in Saccharomyces cerevisiae and equipped with 5' and 3' restriction sites for Golden Gate cloning. The codon-optimized coding sequences for crTDC, acTDC, and cnsBv2 are provided in SEQ ID NOs: 47, 50, and 51, respectively.

[0380] Plasmids pNMT-crTDC, pNMT-acTDC, and pNMT-cnsBv2 were constructed from individual parts using BsaI Golden Gate assembly (Engler et al., 2008, PLoS One, 3:e3647). The plasmids are identical except for the tryptophan decarboxylase gene and contain the following components: the pTDH3 promoter and tENO1 terminator flanking each decarboxylase gene (operably linked to the pTDH3 promoter), an ampicillin resistance gene, a ColE1 origin of replication, a URA3 auxotrophic marker, and homology arms for integration of the expression cassette into the URA3 locus of the S. cerevisiae genome.

[0381] Transformation of S. cerevisiae with pNMT-crTDC, pNMT-acTDC, and pNMT-cnsBv2 yielded strains SC-crTDC, SC-acTDC, and SC-cnsBv2, respectively, each containing a single copy of the relevant tryptophan decarboxylase gene.

[0382] Tryptamine biosynthesis The ability of S. cerevisiae strains SC-crTDC, SC-acTDC, SC-cnsBv1, SC-cnsBv2, SC-asTDC1, and SC-psiD to produce tryptamine was determined. A 1 mL culture of YPD (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) was inoculated with a single colony of S. cerevisiae. A 100 μL sample of the culture medium was taken on day 3 of cultivation. Extraction was performed by adding 200 μL of methanol followed by thorough vortexing. The extract was centrifuged extensively to remove cellular debris.

[0383] LC-MS-based analysis and quantification of tryptamine yield were performed as described in Examples 2 and 4.

[0384] As shown in Figure 16, different PLP-dependent tryptophan carboxylases exhibit varying rates of tryptamine production, with CnsBv2 and asTDC1, which commonly use the TDCs PsiD and CrTDC, being highly efficient.

[0385] [Example 7] Yarrowia containing TDC Plasmid assembly is carried out using the coding sequences for the enzymes essentially as described in Examples 1 and 4-6.

[0386] Yarrowia strain construction and transformation is performed essentially as described in Holkenbrink et al., Biotechnol J. 2018;13(9):e1700543.

[0387] The ability of Yarrowia to produce tryptamine, psilocybin, and psilocin is determined essentially as described in Examples 2 and 4.

[0388] [Example 8] Aspergillus Plasmid assembly is carried out using the coding sequences for the enzymes essentially as described in Examples 1 and 4-6.

[0389] Aspergillus strain construction and transformation is performed essentially as described in Roux and Chooi. Methods Mol Biol. 2022:2489:75-92.

[0390] The ability of Aspergillus to produce tryptamine is determined essentially as described in Example 2.

[0391] The ability of Aspergillus to produce psilocybin and psilocin is determined essentially as described in Examples 2 and 4-6.

[0392] [Example 9] Corynebacterium glutamicum Plasmid assembly is carried out using the coding sequences for the enzymes essentially as described in Examples 1 and 4-6.

[0393] Strain construction and transformation of C. glutamicum was performed essentially as described in Kim et al., 2023, CSBJ; Dunican & Shivnan, 1989, BioTechnology; Ruan et al., 2015, Biotechnology Letters; and Li et al., 2016, J. Microbiol. Methods.

[0394] The ability of C. glutamicum to produce tryptamine, psilocybin, and psilocin is determined essentially as described in Examples 2 and 4.

[0395] [Example 10] Cell-free production of tryptophan derivatives Plasmid assembly is carried out using the coding sequences for the enzymes essentially as described in Examples 1 and 4-6.

[0396] Cell-free production of tryptophan derivatives using enzymes as described herein is carried out essentially as described in Fricke et al., 2020, Biosynthesis; and Blei et al., 2018, Biosynthesis.

[0397] [Table 2]

[0398] [Table 3]

[0399] [Table 4]

[0400] Table 5

[0401] Table 6

[0402] Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 7-5 Table 7-6 Table 7-7 Table 7-8 Table 7-9 Table 7-10 Table 7-11 Table 7-12 Table 7-13 Table 7-14 Table 7-15 Table 7-16 Table 7-17 Table 7-18 Table 7-19 Table 7-20 Table 7-21 Table 7-22

Claims

1. A cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding a pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase, A cell in which the production level of at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that lacks the exogenous polynucleotide or that contains a second polynucleotide encoding a second tryptophan decarboxylase, wherein the second polynucleotide is not the same as the exogenous polynucleotide.

2. 1. A cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding a pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase, wherein the production level of the at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that either lacks the exogenous polynucleotide or comprises a second polynucleotide encoding a second tryptophan decarboxylase, wherein the second polynucleotide is not the same as the exogenous polynucleotide, and the PLP-dependent tryptophan decarboxylase is one of the following: a) GX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ A first amino acid sequence of R, wherein: X １ , X ２ , X ３ , X ４ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ , X １５ , and X １６ is any amino acid residue, X ５ and X ６ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X ７ is an amino acid residue selected from the group consisting of serine and threonine, and X １７ is an amino acid residue selected from the group consisting of alanine and glycine; b) X １ X ２ X ３ X ４ X ５ X ６ HX ７ X ８ X ９ X １０ KX １１ X １２ X １３ X １４ X １５ X １６ a second amino acid sequence of the formula: X １ is any amino acid residue selected from the group consisting of tyrosine, leucine, phenylalanine, valine, cysteine, and tryptophan; X ２ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ and X １５ is any amino acid residue, X ３ is any amino acid residue selected from the group consisting of serine and threonine, and X １６ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; c) DX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ GX １５ X １６ X １７ X １８ X １９ X ２０ X ２１ A third amino acid sequence of D, wherein: X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ , X １５ , X １７ , X １８ , X １９ and X ２１ is any amino acid residue, and X ７ is an amino acid residue selected from the group consisting of proline and asparagine; X １６ is an amino acid residue selected from the group consisting of threonine, proline, and valine, and X ２０ is 1, 2, 3, 4, 5, or 6 amino acid residues, and is any amino acid residue; d) X １ X ２ HX ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ a fourth amino acid sequence, wherein: X １ is an amino acid residue selected from the group consisting of tryptophan and phenylalanine; X ２ , X ３ , X ６ , X ９ and X １０ is any amino acid residue, X ４ is an amino acid residue selected from the group consisting of aspartic acid and glutamic acid; X ５ is an amino acid residue selected from the group consisting of glycine, alanine, and serine; X ７ is an amino acid residue selected from the group consisting of phenylalanine, tyrosine, and tryptophan, and X ８ is an amino acid residue selected from the group consisting of glycine, alanine, serine and threonine; and e) X １ X ２ X ３ X ４ X ５ X ６ X ７ HKX ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ A fifth amino acid sequence of: X １ is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, histidine, and asparagine; X ２ is an amino acid residue selected from the group consisting of serine and threonine; X ３ , X ４ , X ５ , X ７ , X １０ , X １１ , X １２ , X １３ and X １４ is any amino acid residue, X ６ is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, and threonine; X ８ is an amino acid residue selected from the group consisting of tryptophan, phenylalanine, tyrosine, leucine, methionine, and cysteine; X ９ is an amino acid residue selected from the group consisting of leucine, isoleucine, valine, methionine, and phenylalanine; X １５ is an amino acid residue selected from the group consisting of cysteine, alanine and serine, and X １６ is an amino acid residue selected from the group consisting of glycine, serine, alanine and threonine; A cell comprising one or more or all of the above.

3. a) GX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ A first amino acid sequence of R, wherein: X １ , X ２ , X ３ , X ８ , X ９ , X １２ , X １３ , X １４ , X １５ , and X １６ is any amino acid residue, X ４ is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X ５ and X ６ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X ７ is an amino acid residue selected from the group consisting of serine and threonine; X １０ is an amino acid residue selected from the group consisting of asparagine, isoleucine, and valine; X １１ is an amino acid residue selected from the group consisting of leucine, isoleucine, methionine and valine, and X １７ is an amino acid residue selected from the group consisting of alanine and glycine; b) X １ X ２ X ３ X ４ X ５ X ６ HX ７ X ８ X ９ X １０ KX １１ X １２ X １３ X １４ X １５ X １６ a second amino acid sequence of the formula: X １ is any amino acid residue selected from the group consisting of tyrosine, leucine, phenylalanine, valine, cysteine, and tryptophan; X ２ , X ４ , X ５ , X ６ , X ７ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ and X １５ is any amino acid residue, X ３ is any amino acid residue selected from the group consisting of serine and threonine; X ８ is any amino acid residue selected from the group consisting of serine and methionine, and X １６ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; c) DX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ GX １５ X １６ X １７ X １８ X １９ X ２０ X ２１ A third amino acid sequence of D, wherein: X １ , X ２ , X ３ , X ５ , X ６ , X ９ , X １０ , X １１ , X １５ , X １７ and X １８ is any amino acid residue, X ４ is an amino acid residue selected from the group consisting of glycine, alanine, serine, aspartic acid, and asparagine; X ７ is an amino acid residue selected from the group consisting of proline and asparagine; X ８ is an amino acid residue selected from the group consisting of phenylalanine, isoleucine, leucine, threonine, tyrosine, and tryptophan; X １２ is an amino acid residue selected from the group consisting of alanine and glycine; X １３ is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X １４ is an amino acid residue selected from the group consisting of cysteine, alanine, serine, threonine, leucine, glycine and valine, and X １６ is an amino acid residue selected from the group consisting of threonine, proline, and valine; X １９ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X ２０ is 1, 2, 3, 4, 5, or 6 amino acid residues, and is any amino acid residue; and X ２１ is an amino acid residue selected from the group consisting of isoleucine, leucine, valine and threonine; d) X １ X ２ HX ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ a fourth amino acid sequence, wherein: X １ is an amino acid residue selected from the group consisting of tryptophan and phenylalanine; X ２ , X ３ , X ６ and X １０ is any amino acid residue, X ４ is an amino acid residue selected from the group consisting of aspartic acid and glutamic acid; X ５ is an amino acid residue selected from the group consisting of glycine, alanine, and serine; X ７ is an amino acid residue selected from the group consisting of phenylalanine, tyrosine, and tryptophan; X ８ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine, and X ９ is an amino acid residue selected from the group consisting of alanine, glycine, leucine, serine and threonine; and e) X １ X ２ X ３ X ４ X ５ X ６ X ７ HKX ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ A fifth amino acid sequence of: X １ is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, histidine, and asparagine; X ２ is an amino acid residue selected from the group consisting of serine and threonine; X ３ , X ４ , X ５ , X ７ , X １０ , X １２ and X １４ is any amino acid residue, X ６ is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, and threonine; X ８ is an amino acid residue selected from the group consisting of tryptophan, phenylalanine, tyrosine, and cysteine; X ９ is an amino acid residue selected from the group consisting of leucine, isoleucine, valine, methionine, and phenylalanine; X １１ is an amino acid residue selected from the group consisting of glutamine, valine, threonine and alanine; X １３ is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, and valine; X １５ is an amino acid residue selected from the group consisting of cysteine, alanine and serine, and X １６ is an amino acid residue selected from the group consisting of glycine, serine, alanine and threonine; The cell of claim 2.

4. a) GX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ A first amino acid sequence of R, wherein: X １ , X ２ , X ３ , X ８ , X １２ , X １３ , X １４ , X １５ , and X １６ is any amino acid residue, X ４ is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X ５ and X ６ is an amino acid residue selected from the group consisting of glycine and threonine; X ７ is an amino acid residue selected from the group consisting of serine and threonine; X ９ is an amino acid residue selected from the group consisting of alanine, glycine, cysteine, and serine; X １０ is an amino acid residue selected from the group consisting of asparagine, isoleucine, and valine; X １１ is an amino acid residue selected from the group consisting of leucine, methionine and valine, and X １７ is an amino acid residue selected from the group consisting of alanine and glycine; b) X １ X ２ X ３ X ４ X ５ X ６ HX ７ X ８ X ９ X １０ KX １１ X １２ X １３ X １４ X １５ X １６ a second amino acid sequence of the formula: X １ is any amino acid residue selected from the group consisting of tyrosine, leucine, phenylalanine, valine, cysteine, and tryptophan; X ２ , X ４ , X ７ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ and X １５ is any amino acid residue, X ３ is any amino acid residue selected from the group consisting of serine and threonine; X ５ is any amino acid residue selected from the group consisting of glutamine and glutamic acid; X ６ is any amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X ８ is the amino acid residue serine, and X １６ is the amino acid residue glycine; c) DX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ GX １５ X １６ X １７ X １８ X １９ X ２０ X ２１ A third amino acid sequence of D, wherein: X １ , X ２ , X ３ , X ５ , X ６ , X ９ , X １１ , X １５ , X １８ and X ２０ is any amino acid residue, X ４ is an amino acid residue selected from the group consisting of glycine, alanine, serine, aspartic acid, and asparagine; X ７ is the amino acid residue proline, X ８ is an amino acid residue selected from the group consisting of phenylalanine, threonine, and tyrosine; X １０ is an amino acid residue selected from the group consisting of isoleucine, leucine, valine, alanine and glycine; X １２ is an amino acid residue selected from the group consisting of alanine and glycine; X １３ is an amino acid residue selected from the group consisting of serine, threonine, and asparagine; X １４ is an amino acid residue selected from the group consisting of cysteine, serine, threonine, leucine, glycine and valine; X １６ is an amino acid residue selected from the group consisting of threonine, proline, and valine; X １７ is an amino acid residue selected from the group consisting of asparagine, lysine, arginine, aspartic acid, serine, and valine; X １９ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine, and X ２１ is an amino acid residue selected from the group consisting of isoleucine, leucine, valine and threonine; d) X １ X ２ HX ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ a fourth amino acid sequence, wherein: X １ is an amino acid residue selected from the group consisting of tryptophan and phenylalanine; X ２ and X ３ is any amino acid residue, X ４ is the amino acid residue aspartic acid, X ５ is an amino acid residue selected from the group consisting of glycine, alanine, and serine; X ６ is an amino acid residue selected from the group consisting of alanine, valine, and serine; X ７ is an amino acid residue selected from the group consisting of phenylalanine, tyrosine, and tryptophan; X ８ is an amino acid residue selected from the group consisting of glycine, alanine, serine, and threonine; X ９ is an amino acid residue selected from the group consisting of alanine, glycine, leucine, serine, and threonine, and X １０ is an amino acid residue selected from the group consisting of serine, alanine, phenylalanine and threonine; and e) X １ X ２ X ３ X ４ X ５ X ６ X ７ HKX ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ A fifth amino acid sequence of: X １ is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, histidine, and asparagine; X ２ is an amino acid residue selected from the group consisting of serine and threonine; X ３ , X ４ and X １４ is any amino acid residue, X ５ is the amino acid residue tryptophan, X ６ is the amino acid residue aspartic acid, X ７ is an amino acid residue selected from the group consisting of alanine and glycine; X ８ is the amino acid residue tryptophan, X ９ is the amino acid residue leucine, X １０ is an amino acid residue selected from the group consisting of phenylalanine, leucine, glycine, asparagine, and methionine; X １１ is the amino acid residue glutamine, X １２ is an amino acid residue selected from the group consisting of threonine, serine, leucine, isoleucine, valine, and alanine; X １３ is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, and valine; X １５ is an amino acid residue selected from the group consisting of cysteine ​​and serine, and X １６ is an amino acid residue selected from the group consisting of glycine and serine; The cell of claim 3.

5. The cell of any one of claims 2 to 4, wherein the exogenous polynucleotide comprises, in 5' to 3' order, a first amino acid sequence, a second amino acid sequence, a third amino acid sequence, a fourth amino acid sequence, and a fifth amino acid sequence.

6. 6. The cell of claim 5, wherein there are about 2 to about 50 amino acids, or about 10 to about 35 amino acids, or about 12 to about 25 amino acids between the first amino acid sequence and the second amino acid sequence.

7. 7. The cell of claim 5 or 6, wherein there are about 2 to about 50 amino acids, or about 20 to about 40 amino acids, or about 25 to about 35 amino acids between the second amino acid sequence and the third amino acid sequence.

8. 8. The cell of claim 5, wherein there are about 5 to about 50, or about 10 to about 25, or about 12 to about 22 amino acids between the third amino acid sequence and the fourth amino acid sequence.

9. 9. The cell of claim 5, wherein there are about 5 to about 50 amino acids, or about 10 to about 30, or about 15 to about 20 amino acids between the fourth amino acid sequence and the fifth amino acid sequence.

10. A cell capable of producing at least one tryptophan derivative, comprising at least one exogenous polynucleotide encoding an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29 and 48, or a sequence having at least about 70% sequence identity thereto, A cell in which the production level of at least one tryptophan derivative in the cell is increased compared to the production level of a second tryptophan derivative in an equivalent cell that lacks the exogenous polynucleotide or that contains a second polynucleotide encoding a second tryptophan decarboxylase, wherein the second polynucleotide is not the same as the exogenous polynucleotide.

11. The cell of claim 10, wherein the exogenous polynucleotide encodes the amino acid sequence shown in SEQ ID NO:

1.

12. The cell of claim 10, wherein the exogenous polynucleotide encodes the amino acid sequence shown in SEQ ID NO:

7.

13. The cell of claim 10, wherein the exogenous polynucleotide encodes the amino acid sequence shown in SEQ ID NO:

9.

14. 14. The cell of any one of claims 1 to 13, wherein the PLP-independent tryptophan decarboxylase is a PsiD tryptophan decarboxylase, and optionally comprises the amino acid sequence shown in SEQ ID NO:

9.

15. The cell of any one of claims 1 to 14, wherein the cell comprises two or more copies of the exogenous polynucleotide.

16. The cell of any one of claims 1 to 15, wherein the exogenous polynucleotide is operably linked to a heterologous promoter.

17. The cell of any one of claims 1 to 16, further comprising a polynucleotide encoding an additional tryptophan decarboxylase.

18. 18. The cell of claim 17, wherein the additional tryptophan decarboxylase is PsiD, optionally comprising the amino acid sequence set forth in SEQ ID NO: 9 or a sequence having at least about 70% sequence identity thereto.

19. The cell of any one of claims 1 to 18, wherein the cell further comprises a polynucleotide encoding tryptamine 4-monooxygenase.

20. 20. The cell of claim 19, wherein the tryptamine 4-monooxygenase is PsiH optionally comprising the amino acid sequence set forth in SEQ ID NO: 11 or a sequence having at least about 70% sequence identity thereto.

21. 21. The cell of claim 20, wherein the cell comprises two or more copies of a polynucleotide encoding PsiH.

22. The cell of any one of claims 1 to 21, wherein the cell further comprises a polynucleotide encoding 4-hydroxytryptamine kinase.

23. 23. The cell of claim 22, wherein the 4-hydroxytryptamine kinase is PsiK, optionally comprising the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least about 70% sequence identity thereto.

24. The cell of claim 23 , wherein the cell comprises two or more copies of a polynucleotide encoding PsiK.

25. The cell of any one of claims 1 to 24, wherein the cell further comprises a polynucleotide encoding a methyltransferase.

26. Methyltransferases: A PsiM optionally comprising the amino acid sequence set forth in SEQ ID NO: 15 or a sequence having at least about 70% sequence identity thereto; or RmNMT optionally comprising the amino acid sequence set forth in SEQ ID NO: 42 or a sequence having at least about 70% sequence identity thereto. The cell of claim 25,

27. 27. The cell of claim 26, wherein the cell comprises at least two polynucleotides encoding PsiM and / or RmNMT.

28. The cell of any one of claims 1 to 27, wherein the cell further comprises a polynucleotide encoding a cytochrome P450 reductase.

29. Cytochrome P450 reductase: PcCpr optionally comprising the amino acid sequence set forth in SEQ ID NO: 17 or a sequence having at least about 70% sequence identity thereto; or OsCPR optionally comprising the amino acid sequence set forth in SEQ ID NO: 39 or a sequence having at least about 70% sequence identity thereto. The cell of claim 28,

30. 30. The cell of claim 29, wherein the cell comprises at least two polynucleotides encoding PcCpr and / or OsCPR.

31. 31. The cell of any one of claims 1 to 30, wherein the cell further comprises at least one copy of a polynucleotide encoding an adenosylhomocysteinase optionally comprising the amino acid sequence set forth in SEQ ID NO: 19 or a sequence having at least about 70% sequence identity thereto.

32. 32. The cell of any one of claims 1 to 31, wherein the cell further comprises at least one exogenous polynucleotide encoding an adenosine kinase optionally comprising the amino acid sequence set forth in SEQ ID NO: 21 or a sequence having at least about 70% sequence identity thereto.

33. 32. The cell of any one of claims 1 to 31, wherein the cell further comprises at least one exogenous polynucleotide encoding tryptamine 5-hydroxylase, optionally comprising the amino acid sequence set forth in SEQ ID NO: 36, or a sequence having at least about 70% sequence identity thereto.

34. The cell of any one of claims 1 to 33, wherein the cell is a yeast cell.

35. 35. The cell of claim 34, wherein the yeast cell is a Saccharomyces species.

36. 36. The cell of claim 35, wherein the Saccharomyces species is S. cerevisiae.

37. 37. The cell of claim 36, wherein the yeast cell is a Yarrowia species.

38. The cell of any one of claims 1 to 33, wherein the cell is a bacterial cell.

39. 39. The cell of claim 38, wherein the bacterial cell is an Escherichia species or a Corynebacterium species.

40. 40. The cell of claim 39, wherein the Escherichia species is Escherichia coli.

41. 41. The cell of claim 40, wherein the Corynebacterium species is C. glutamicum.

42. The cell of any one of claims 1 to 33, wherein the cell is a fungal cell.

43. 43. The cell of claim 42, wherein the fungal cell is an Aspergillus species.

44. 44. The cell of any one of claims 1 to 43, wherein the at least one tryptophan derivative is tryptamine or a tryptamine-derived alkaloid.

45. The cell of any one of claims 1 to 44, wherein the tryptophan derivative is tryptamine.

46. 46. ​​The cell of claim 45, wherein the tryptamine-derived alkaloid is psilocybin.

47. a) at least one exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase as defined in any one of claims 1 to 10; b) at least one exogenous polynucleotide encoding a PsiH tryptamine 4-monooxygenase; c) at least one exogenous polynucleotide encoding a PsiK hydroxytryptamine kinase; and d) at least one exogenous polynucleotide encoding a PsiM methyltransferase. A cell capable of producing at least one tryptophan derivative, comprising:

48. a) at least one exogenous polynucleotide encoding a PLP-dependent tryptophan decarboxylase having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29, and 48, or a sequence having at least about 70% sequence identity thereto; b) at least one exogenous polynucleotide encoding a PsiH tryptamine 4-monooxygenase; c) at least one exogenous polynucleotide encoding a PsiK hydroxytryptamine kinase; and d) at least one exogenous polynucleotide encoding a PsiM methyltransferase. A cell capable of producing at least one tryptophan derivative, comprising:

49. 50. The cell of claim 48 or 49, wherein the cell is capable of an increased level of production of at least one tryptophan derivative compared to the level of production of the at least one tryptophan derivative in an equivalent cell comprising a second polynucleotide encoding a second tryptophan decarboxylase, the second polynucleotide being different from (a), and optionally the second polynucleotide encoding a PsiD tryptophan decarboxylase.

50. 50. The cell of claim 48 or 49, wherein the tryptophan derivative is tryptamine or psilocybin.

51. 51. The cell of any one of claims 47 to 50, wherein the cell further comprises at least one exogenous polynucleotide encoding a cytochrome P450 reductase, and optionally, the cytochrome P450 reductase is a PcCpr cytochrome P450 reductase.

52. 52. The cell of any one of claims 47 to 51, wherein the cell further comprises at least one exogenous polynucleotide encoding an adenosylhomocysteinase, and optionally, the adenosylhomocysteinase is Sah1 adenosylhomocysteinase.

53. 53. The cell of any one of claims 47 to 52, wherein the cell further comprises at least one polynucleotide encoding an adenosine kinase, optionally Ado1 adenosine kinase.

54. 54. The cell of any one of claims 47 to 53, wherein the cell further comprises at least one polynucleotide encoding an additional tryptophan decarboxylase, and optionally the additional tryptophan decarboxylase is PsiD and / or CrTDC tryptophan decarboxylase.

55. 11. A vector comprising a polynucleotide encoding a PLP-dependent tryptophan decarboxylase as defined in any one of claims 1 to 10, optionally wherein the polynucleotide is operably linked to a heterologous promoter capable of directing expression of the polynucleotide.

56. A vector comprising a polynucleotide encoding a PLP-dependent tryptophan decarboxylase having the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29 and 48, wherein optionally the polynucleotide is operably linked to a heterologous promoter capable of directing expression of the polynucleotide in a cell that does not naturally express the polynucleotide.

57. 57. A host cell comprising the vector of claim 55 or 56.

58. 60. A method for producing at least one tryptophan derivative, comprising culturing a cell according to any one of claims 1 to 54 and 57 under conditions suitable for the production of at least one tryptophan derivative.

59. 59. The method of claim 58, further comprising extracting at least one tryptophan derivative from the cells.

60. 60. The method of claim 59, wherein the tryptophan derivative extracted is tryptamine or a tryptamine-derived alkaloid.

61. 61. The method of claim 60, wherein the tryptophan derivative is tryptamine.

62. 61. The method of claim 60, wherein the tryptamine-derived alkaloid is psilocybin.

63. 11. A method for producing at least one tryptophan derivative, comprising contacting tryptophan with at least one pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase as defined in any one of claims 1 to 10 under conditions suitable for the production of at least one tryptophan derivative.

64. A method for producing at least one tryptophan derivative, comprising contacting tryptophan with at least one pyridoxal phosphate (PLP)-dependent tryptophan decarboxylase comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 29 and 48, or a sequence having at least about 70% sequence identity thereto, under conditions suitable for the production of at least one tryptophan derivative.

65. 65. The method of claim 63 or 64, wherein the method is carried out in a bioreactor.

66. A tryptophan derivative produced by the method according to any one of claims 58 to 65.

67. 68. A composition comprising the cells of any one of claims 1 to 54 and 57, or the extracted tryptophan derivative of claim 67.

68. 68. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject the composition of claim 67.

69. 69. The method of claim 68, wherein the disease or disorder is a neurodevelopmental, neurological, neuromuscular, movement, psychiatric, or psychological disease, disorder, or syndrome.

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