WO2023081842A2

WO2023081842A2 - Alternative biosynthesis pathways for the production of psilocybin and intermediates or side products

Info

Publication number: WO2023081842A2
Application number: PCT/US2022/079329
Authority: WO
Inventors: John Andrew Jones; Fiona KANIS
Original assignee: Miami University; University of Miami
Current assignee: Miami University; University of Miami
Priority date: 2021-11-05
Filing date: 2022-11-04
Publication date: 2023-05-11
Anticipated expiration: 2024-05-05
Also published as: WO2023081842A3

Abstract

Provided are methods, prokaryotic host cells, expression vectors, and kits for the production of psilocybin or an intermediate or a side product thereof. In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.

Description

AL TERNATIVE BIOSYNTHESIS PATHWAYS FOR THE PRODUCTION OF PSILOCYBIN AND INTERMEDIATES OR SIDE PRODUCTS

FIELD

[0001] The general inventive concepts relate to the field of medical therapeutics and more particularly to alternative biosynthesis pathways for the production of psilocybin and intermediates or side products.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The instant application is entitled to priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63/263,618, filed November 5, 2021, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (315691-00045.xml; Size: 45,258 bytes; and Date of Creation: November 4, 2022) is herein incorporated by reference in its entirety.

BACKGROUND

[0004] Psilocybin (4-phosphoryloxy-N. AAlmicthyltryptaminc) has gained attention in pharmaceutical markets as a result of recent clinical studies. The efficacy of psilocybin has been demonstrated for the treatment of anxiety in terminal cancer patients and alleviating the symptoms of post-traumatic stress disorder (PTSD). Most recently, the FDA has approved the first Phase lib clinical trial for the use of psilocybin as a treatment for depression that is not well controlled with currently available interventions such as antidepressants and cognitive behavioral therapies.

[0005] The biosynthesis of psilocybin has been of much interest over the past few years, however, all published studies have focused on the use of the psilocybin biosynthesis pathway, PsiDHKM, originally reported by the Hoffineister group (Fricke et al., 2017), with only minor variations in gene source or pathway construction (Adams et al., 2019; Bao et al., 2021; Hoefgen et al., 2018; Milne et al., 2020). The dimethylation of the terminal amine by PsiM is key to psychedelic properties of psilocybin with the pathway intermediate, baeocystin, having been reported to not elicit a head twitch response in a rodent model (Sherwood et al., 2020). Additionally, the activity of PsiM has only been reported on 4- phosphoryloxytryptamines, limiting substrates available for methylation using the established pathway (Fricke et al., 2019, 2017). Due to the important of this dimethylation for bioactivity, there exists a desire to identify alternative enzymatic routes to accomplish this methyl addition on a variety of tryptamine substrates.

[0006] There remains a need for methods for the production of psilocybin and intermediates or side products thereof employing alternative biosynthetic pathways.

SUMMARY

[0007] Provided is a method for the production of psilocybin or an intermediate or a side product thereof comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of TrpM, psiD, psiK, Tdc, and combinations thereof; and culturing the host cell. In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus , and Streptomyces venezuelae.

[0008] In some embodiments, the intermediate or side product of psilocybin is 4- hydroxytryptophan, 4-hydroxy-A-methyltryptophan, 4-hydroxy-A A-dimethyltryptophan, 4- hydroxy-A N, A-tri methyl tryptophan. 4-hydroxy-A-methyltryptamine (norpsilocin), 4- hydroxy-AA-di methyl tryptamine (psilocin), 4-hydroxy-N,N,N-trimethyltryptamine (4-OH- TMT), A-methyltryptamine (NMT), A,A-dimethyltryptamine (DMT), or N,N,N- trimethyltryptamine (TMT). In some embodiments the intermediate of psilocybin is 4- hydroxytryptophan, 4-hydroxy-A-methyltryptophan, 4-hydroxy-A A-dimethyltryptophan, or psilocin. In some embodiments, the side product of psilocybin is 4-hydroxy-A, A, A- trimethyltryptophan, 4-hydroxy-A-methyltryptamine (norpsilocin), 4-hydroxytryptamine, or 4-hydroxy-N,N,N -trimethyltryptamine (4-OH-TMT) .

[0009] Also provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of TrpM, psiD, psiK, Tdc, and combinations thereof. Provided is a vector for introducing at least one gene; the gene may be selected from: TrpM, psiD, psiK, Tdc, and combinations thereof. Also provided is a transfection kit comprising an expression vector as described herein.

DESCRIPTION OF THE FIGURES

[0010] FIG. 1 shows a pathway for production of methylated tryptophans and methylated tryptamines consisting of the enzymes TrpM and psiD. TrpM: methyltransferase, psiD: tryptophan decarboxylase, Tdc: tryptophan decarboxylase.

[0011] FIG. 2 illustrates the evaluation of TrpM activity on tryptophan in E. coli. Temperatures of 25, 30, and 37°C were tested to identify optimal production. The products being evaluated were mono-, di-, and tri-A-mcthylatcd tryptophan. Taken together, TrpM activity was found to be highest at 25°C.

[0012] FIG. 3 shows a biosynthesis pathway for psilocybin (Adams, et al., 2019), tracking first down and then to the right, characterized by initial activity of PsiD and PsiK, followed by PsiM, and the alternative psilocybin pathway proposed in this study, tracking first to the right, then down, characterized by initial activity of TrpM, followed by Tdc or PsiD and PsiK. TrpM acts on 4-hydroxytyptophan to produce mono- and di-methylated 4- hydroxytryptophan. The production of N, N, A-trimethyl-4-hydroxytryptophan is not observed as indicated by the X.

[0013] FIG 4 shows production of A-methylated 4-hydroxytryptophan tested at different temperatures. Production of methylated 4-hydroxytryptophan surpassed that of methylated tryptophan shown in FIG. 2 and production of N, A-di methyl -4-hydroxytryptophan was higher than that of A-methyl-4-hydroxytryptophan. Optimal temperature was 30°C.

[0014] FIG. 5 is an HPLC graph (A280) showing each product of the chemical synthesis of methylated tryptophan. The top line represents the combined methylated products before preparative HPLC purification. The second line represents Fraction 2, a mixture of tryptophan (m/z 205) and A-methyltryptophan (m/z 219). The third line represents Fraction 3, a mixture of A-methyltryptophan (m/z 219), N, A-dimethyltryptophan (m/z 233), and N,N,N- trimethyltryptophan (m/z 247). The bottom line represents Fraction 4, a mixture of N,N- dimethyltryptophan (m/z 233), and N, N, A-trimethyltryptophan (m/z 247).

[0015] FIGs. 6A-6B show LCMS extracted ion chromatograms. FIG. 6A shows an LCMS extracted ion chromatogram showing evidence of NMT (m/z 175) production from methylated tryptophan by psiD. Each of the arrows indicate peaks with the correct NMT mass. FIG. 6B shows an LCMS extracted ion chromatogram showing evidence of DMT (m/z 189) production from methylated tryptophan by psiD. Each arrow indicates peaks with the correct DMT mass.

[0016] FIGs. 7A-7C show an evaluation of psiD activity on methylated tryptophan fractions. FIG. 7A: Fraction 2. FIG. 7B: Fraction 3. FIG. 7C: Fraction 4. Products shown are based on methylated tryptophans present in respective fractions. * Trimethylated product was not observed. Error bars represent +/- 1 standard deviation from the mean of duplicate samples.

[0017] FIGs. 8A-8C show extracted ion chromatograms with accurate mass MS and MS/MS for 4-hydroxytryptophan (FIG. 8A), A-methyl-4-hydroxytryptophan (FIG. 8B), and N,N- dimethyl-4-hydroxytryptophan (FIG. 8C).

[0018] FIGs. 9A-9C show evaluation of alkaline phosphatase denaturation method and activity on psilocybin. FIG. 9A: No AP enzyme. FIG. 9B: AP enzyme without being subjected to heat denaturation. FIG. 9C: AP enzyme subjected to heat denaturation. *no psilocin detected; #no psilocybin detected.

[0019] FIG. 10 shows evaluation of in vivo PsiK activity on psilocin fed following AP reaction. Error bars represent +/- 1 standard deviation from the mean of duplicate samples.

DETAILED DESCRIPTION

[0020] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.

[0021] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a cell” means one cell or more than one cell. [0023] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0024] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or “consisting essentially of’ such features, and vice-versa.

[0025] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range in explicitly recited.

[0026] As used herein, the term “prokaryotic host cell” means a prokaryotic cell that is susceptible to transformation, transfection, transduction, or the like, with a nucleic acid construct or expression vector comprising a polynucleotide. The term “prokaryotic host cell” encompasses any progeny that is not identical due to mutations that occur during replication.

[0027] As used herein, the term “recombinant cell” or “recombinant host” means a cell or host cell that has been genetically modified or altered to comprise a nucleic acid sequence that is not native to the cell or host cell. In some embodiments the genetic modification comprises integrating the polynucleotide in the genome of the host cell. In further embodiments the polynucleotide is exogenous in the host cell.

[0028] As used herein, the term “intermediate” of psilocybin means an intermediate in the production or biosynthesis of psilocybin, e.g., 4-hydroxytryptophan, 4-hydroxy-A- methyltryptophan, 4-hydroxy-A. A-dimethyltryptophan, or psilocin.

[0029] As used herein, the term “side product” of psilocybin means a side product in the production or biosynthesis of psilocybin, e.g., 4-hydroxy-A. A. A-trimcthyltryptophan. 4- hydroxy-A-methyltryptamine (norpsilocin), 4-hydroxytryptamine, or 4-hydroxy-N,N,N- trimethyltryptamine (4-OH-TMT). [0030] The materials, compositions, and methods described herein are intended to be used to provide novel routes for the production of psilocybin and intermediates or side products.

I. Methods, vectors, host cells and kits for the production of psilocybin or an intermediate or a side product thereof

Methods

[0031] Provided is a method for the production of psilocybin or an intermediate or a side product thereof. The method comprises contacting a host cell with at least one gene selected from: TrpM, psiD, psiK, Tdc, and combinations thereof to form a recombinant cell; culturing the recombinant cell; and obtaining psilocybin. In certain embodiments, the host cell is a prokaryotic cell. In certain exemplary embodiments, the host cell is an E. coli cell.

[0032] Provided is a method for the production of psilocybin or an intermediate or a side product thereof comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of TrpM, psiD, psiK, Tdc, and combinations thereof; and culturing the host cell. In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus , and Streptomyces venezuelae.

[0033] In certain embodiments, the TrpM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the TrpM gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number MH423322 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the TrpM is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0034] In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 2, 3, 4, 5, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0035] In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number KY984099. 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0036] In certain embodiments, the Tdc gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 19, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the Tdc gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number M25151 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the Tdc is encoded by a nucleotide sequence comprising SEQ ID NO: 8, 9, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0037] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.

[0038] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

[0039] It is envisaged that any intermediate or side product of psilocybin may be produced by any of the methods described herein. In some embodiments, the intermediate or side product of psilocybin is 4-hydroxytryptophan, 4-hydroxy-A-methyltryptophan, 4-hydroxy-A, N- dimethyltryptophan, 4-hydroxy-A, N, A-trimethyltryptophan, 4-hydroxy-A-methyltryptamine (norpsilocin), 4-hydroxy-AA-dimethyltryptamine (psilocin), 4-hydroxy-N,N,N- trimethyltryptamine (4-OH-TMT), A-methyltryptamine (NMT), A, A-dimethyltryptamine (DMT), or AAA-trimethyltryptamine (TMT). In some embodiments the intermediate of psilocybin is 4-hydroxytryptophan, 4-hydroxy-A-methyltryptophan, 4-hydroxy-A, A- dimethyltryptophan, or psilocin. In some embodiments, the side product of psilocybin is 4- hydroxy-AAA-trimethyltryptophan, 4-hydroxy-A-methyltryptamine (norpsilocin), 4- hydroxytryptamine, or 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT).

[0040] In certain embodiments, the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4- hydroxytryptophan, 4-hydroxytryptamine, tryptophan, tryptamine, N-methyltryptophan, N,N- dimethyltryptophan and combinations thereof. In certain exemplary embodiments, the supplement is fed continuously to the host cell. In further embodiments, the host cell is grown in an actively growing culture. Continuous feeding is accomplished by using a series of syringe and/or peristaltic pumps whose outlet flow is directly connected to the bioreactor. The set point of these supplement addition pumps is adjusted in response to real-time measurement of cell biomass and specific metabolic levels using UV-vis absorption and HPLC analysis, respectively. The fed-batch fermentation process is focused on maximizing production of target metabolites through harnessing the ability of an actively growing and replicating cell culture to regenerate key co-factors and precursors which are critical to the biosynthesis of target metabolites. This process notably does not involve the centrifugal concentration and reconstitution of cell biomass to artificially higher cell density and/or into production media that was not used to build the initial biomass. The production process involves the inoculation of the reactor from an overnight preculture at low optical density, followed by exponential phase growth entering into a fed-batch phase of production, culminating in a high cell density culture.

[0041] The psilocybin and intermediate or side products are found extracellularly in the fermentation broth. In certain embodiments, the psilocybin and intermediate or side products are isolated. These target products can be collected through drying the fermentation broth after centrifugation to remove the cell biomass. The resulting dry product can be extracted to further purify the target compounds. Alternatively, the products can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions psilocybin or any of the intermediate or side products into the organic phase. Furthermore, contaminants from the fermentation broth can be removed through extraction leaving the psilocybin and/or intermediate or side products in the aqueous phase for collection after drying or crystallization procedures.

[0042] In certain embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 50 mg/L. In some embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 20 mg/L. In yet further embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 10 mg/L. In certain embodiments, the methods described herein result in a titer of psilocybin of about 1.0 to about 10 mg/L. In further embodiments, the methods described herein result in a titer of psilocybin of about 5 to about 10 mg/L.

[0043] In certain embodiments, the methods described herein result in a molar yield of psilocybin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of psilocybin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of psilocybin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of psilocybin of about 40% to about 60%. In further embodiments, the methods described herein result in a molar yield of psilocybin of about 50%.

Recombinant prokaryotic cells for the production of psilocybin or an intermediate or a side product thereof

[0044] Provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of TrpM, psiD, psiK, Tdc, and combinations thereof.

[0045] In certain embodiments, the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus , and Streptomyces venezuelae.

[0046] In certain embodiments, the TrpM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the TrpM gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number MH423322 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the TrpM is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. [0047] In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 2, 3, 4, 5, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0048] In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number KY984099. 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0049] In certain embodiments, the Tdc gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 19, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the Tdc gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number M25151 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the Tdc is encoded by a nucleotide sequence comprising SEQ ID NO: 8, 9, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. [0050] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.

[0051] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

Expression vectors

[0052] Provided is a vector for introducing at least one gene; the gene may be selected from: TrpM, psiD, psiK, Tdc, and combinations thereof.

[0053] In certain embodiments, the TrpM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the TrpM gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number MH423322 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the TrpM is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0054] In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 2, 3, 4, 5, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0055] In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0056] In certain embodiments, the Tdc gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 19, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the Tdc gene encodes a polypeptide comprising the amino acid sequence of Genbank accession number M25151 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the Tdc is encoded by a nucleotide sequence comprising SEQ ID NO: 8, 9, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0057] In certain embodiments, the expression vector comprises a TrpM gene, a psiD gene, and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the expression vector comprises a TrpM gene, a Tdc gene, and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the expression vector comprises a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the expression vector comprises a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.

[0058] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

Kits

[0059] Provided is a transfection kit comprising an expression vector as described herein. Such a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes. Each of such container means comprises components or a mixture of components needed to perform a transfection. Such kits may include, for example, one or more components selected from vectors, cells, reagents, lipid-aggregate forming compounds, transfection enhancers, or biologically active molecules.

EXAMPLES

Materials and Methods

Bacterial strains, vectors, and media

[0060] E. coli DH5a was used for all molecular cloning, while E. coli BL21 star™ (DE3) was used for fermentation experiments. Plasmid transformations followed standard chemical competent bacterial transformation protocols. Cultures for production experiments used Andrew's Magic Media (AMM) (He et al ,, 2015), and all cloning and cell propagation was performed using Luria Broth (LB), When using pETM6-SDM2x based vectors (Adams et al., 2019), ampicillin (80gg/mL) was used for plasmid retention, lire plasmid pFB06, containing the sequence for TrpM, was gifted by the Hoffineister group (Blei et al., 2018).

[0061] Plasmid construction: pFB06 containing TrpM was restriction enzyme digested with Ndel and Xho\. gel extracted, and ligated into a similarly digested pETM6-SDM2x plasmid backbone, resulting in pETM6-SDM2x-TrpM.

[0062] Standard screening conditions: Screening was done in 48-well plates with a 2 mL culture volume at 37 °C. AMM supplemented with 4-hydroxyindole (50 mg/L), serine (1 g/L), and methionine (5 g/L) and ampicillin were used unless stated otherwise. Overnight cultures were grown from a freezer stock culture in AMM with ampicillin and ammo acid supplements for 12-16 hours in a shaking 37 °C incubator. Induction with 1 mM isopropyl [3- D-l “thiogalactopyranoside (IPTG) occurred 2 hours after inoculation, unless stated otherwise. After 24 hours of growth, HPLC and LCMS analysis was performed as described in the analytical methods below.

[0063] Fermentation Optimization: A variety of conditions were tested on our TrpM vector to enhance production of methylated tryptophan and methylated 4-hydroxytryptophan. The effect of varying induction times was tested, as well as temperature (25 °C, 30 °C, 47 °C, 42 °C), base media (AMM, LB), concentration of supplements: tryptophan, serine, and methionine (0 g/L, 1 g/L, 5 g/L), and concentration of 4-hydroxyindole feed (50 mg/L, 100 mg/L). All screening was conducted in 48-well plates using standard screening conditions unless stated otherwise.

[0064] Chemical Synthesis of Methylated Tryptophan: Tryptophan (4.9 mmol, 1.0 g) was suspended in MeOH (0.25 mol, 10.0 mL) and vortexed for 10 seconds until tryptophan was completely dissolved. The solution was placed in a 120 mL round-bottomed flask with a stir bar, followed by the addition of methyl iodide (35 mmol, 5.0 g). The mixture was allowed to stir for approximately 30 seconds. A liquid cesium hydroxide solution (50 wt% in water) was added to the reaction mixture (105 mmol CsOH). The round-bottomed flask was capped, covered with foil, and stirred overnight. The next day significant precipitant was present. The pH reaction products were reduced to pH = 5 with the addition of acetic acid, prior to purification by preparative HPLC. [0065] Separation and Purification of Methylated Tryptophan: The methylated tryptophans mixture was purified by preparative HPLC using a Thermo Scientific Ultimate 3000 High- Performance Liquid Chromatography (HPLC) system equipped with Diode Array Detector (DAD) and an Agilent Polaris C18-A column (250 mm x 21.2 mm, 5 pm) with mobile phases of water (A) and acetonitrile (B), both containing 0.1% formic acid starting at a flow rate of 5 mL/min and switching to a flow rate of 10 mL/min at 0.9 min: 0 min, 5% B; 0.9 min, 5% B; 10 min, 40% B; 11.25 min, 100% B; 14.5 min, 100% B; 14.5 min, 5% B; 17 min, 5% B.

Injections of 1000 pL were used, resulting in three peaks with retention times of approximately 11.9, 12.2, and 12.9 minutes. The fraction collection was triggered by peak height in the UV absorbance spectrum at 280 nm. The three fractions were analyzed using a Thermo Scientific Ultimate 3000 High-Performance Liquid Chromatography (HPLC) system equipped with Diode Array Detector (DAD) and Thermo Scientific ISQ™ EC single quadrupole mass spectrometer (MS) as presented above. The collected fractions were then dried under vacuum prior to use.

[0066] Methylated Tryptophan Supplementation into AMM: Once the fractions of methylated tryptophan were separated and purified, they were evaporated with a rotary evaporator and lyophilized to obtain a solid product that could be supplemented into AMM media and fed to pETM6-SDM2x-psiD. Due to fraction 1 being mostly unreacted tryptophan, only fractions 2, 3, and 4 were used. The final weights obtained for each of the fractions 2, 3, and 4 were 0.1 g, 0.43 g, and 0.21 g respectively. Stock solutions were made from each of the three fractions in MilliQ H2O so they could be supplemented into AMM at a final concentration of 50 mg/L.

[0067] Analytical Methods: Primary metabolite analysis was performed on a Thermo Scientific Ultimate 3000 High-Performance Liquid Chromatography (HPLC) system equipped with Diode Array Detector (DAD) and Thermo Scientific ISQ™ EC single quadrupole mass spectrometer (MS). Samples were prepared for HPLC and LC-MS analysis by centrifugation at 15,000 x g for 5 min. A volume of 2 pL of the resulting supernatant was then injected for analysis.

[0068] Quantification of aromatic metabolites was performed using absorbance at 280 nm from the DAD or using extracted ion chromatographs derived from the total ion chromatograph. The metabolites were separated using an Agilent Zorbax Eclipse XDB-C18 analytical column (3.0 mm x 250 mm, 5 pm) with mobile phases of water (A) and acetonitrile (B) both containing 0.1% formic acid at a rate of 1 mL/min: 0 min, 5% B; 0.43 min, 5% B; 5.15 min, 19% B; 6.44 min, 100% B; 7.73 min, 100% B; 7.73 min, 5% B; 9.87 min, 5% B.

[0069] When MS analysis was used, analytes were measured in positive ion mode at the flow rate, solvent gradient, and column conditions described above. The instrument was equipped with a heated electrospray ionization (HESI) source and supplied > 99% purity nitrogen from a Peak Scientific Genius XE 35 laboratory nitrogen generator. The source and detector conditions were as follows: sheath gas pressure of 80.0 psig, auxiliary gas pressure of 9.7 psig, sweep gas pressure of 0.5 psig, foreline vacuum pump pressure of 1.55 Torr, vaporizer temperature of 500 °C, ion transfer tube temperature of 300 °C, source voltage of 3049 V, and source current of 15.90 pA.

[0070] High Resolution Liquid Chromatography Mass Spectrometry (LC-MS) and Mass Spectrometry-Mass Spectrometry (LC-MS/MS) data were measured on a Thermo Scientific LTQ Orbitrap XL mass spectrometer equipped with an Ion Max ESI source using the same mobile phases and column described above. The flow rate was adjusted to 0.250 mL/min resulting in a method with the following gradient: 0 min, 5% A; 1 min, 5% A; 24 min, 19% A; 30 min, 100 % A; 36 min 100% A; 36 min, 5% A; 46 min, 5% A.

[0071] Orbitrap mass spectrometry parameters in positive mode were spray voltage 3.5 kV, capillary temperature 275 °C, capillary voltage 23 V and tube lens voltage 80 V (optimized by tuning on 5 -hydroxytryptamine), nitrogen sheath, auxiliary, and sweep gas were 15, 30, 1 a.u., full scan mode (m/z 100-500) at a resolution of 60,000 and an AGC target of le6.

[0072] LC-MS/MS data was collected in the data-dependent acquisition mode, where the full MS scan was followed by fragmentation of the most abundant peak by higher energy collisional dissociation (HCD). Data was collected in the Orbitrap with a minimum m/z of 50 at 30,000 resolution, AGC target of le5, and intensity threshold of 200K using normalized collision energy of 40, default charge state of 1, activation time of 30 ms, and maximum injection times of 200 msec for both MS and MS/MS scans. All data were processed using Xcalibur/Qual Browser 2.1.0 SP1 build (Thermo Scientific). MS/MS fragmentation data can be found in figure 8.

Example 1: Evaluation of TrpM Activity in E. coli [0073] The gene encoding tryptophan methyltransferase, TrpM, from Psilocybe serbica was cloned into the pETM6-SDM2x plasmid vector and expressed in E. coli under the control of the IPTG-inducible T7 promoter system. Functional activity towards the native amino acid substrate, tryptophan, was first evaluated through a small scale, well plate-based assay resulting in mono-, di-, and tri-methylations of the terminal amine (Figure 1). LCMS analysis of the culture media confirmed the presence of low levels of all three of the expected methylated products with retention times and mass-to-charge ratios consistent with a chemically synthesized product. This activity is consistent with the reported activity from an in vitro assay previously reported (Blei et al., 2018).

Example 2: Fermentation Optimization to Enhance TrpM Activity

[0074] After initial screening of our pETM6-SDM2x-TrpM vector showed production of mono-, di-, and tri-methylated tryptophan, the strain underwent various experiments to identify the conditions resulting in the best production. Previous studies evaluated TrpM activity in vitro at 25°C and this served as a starting point for our analysis (Blei et al., 2018). Temperatures of 25, 30, and 37°C were evaluated to see the effect on methylated tryptophan production in the in vivo bacterial system described herein. These experiments were conducted in 125 mb Erlenmeyer flasks containing 25 mb of culture media. These studies show that a fermentation temperature of 25°C generally resulted in the highest production of methylated tryptophans with the most significant differences being noted between the mono- and di-methylated components, with peak production of 2.48 +/- 0.02 mg/L of/V- methyltryptophan, 0.49 +/- 0.02 mg/L N, /V-dimethyltryptophan, and 0.96 +/- 0.06 mg/L /V, N, A'-tri methyl tryptophan (Figure 2).

[0075] Next, the sensitivity to induction timing was evaluated under the top conditions. The chemical inducer, IPTG, was added at 0, 2, and 4 hours post inoculation. Results indicated no significant differences between the conditions tested. An early log phase induction point of 2 hours post inoculation was selected moving forward. No production of methylated tryptophan products was detected in the non-induced samples.

[0076] Finally, the addition of fed substrates, tryptophan and methionine, was used in these experiments to help maximize production. The concentrations of 1 g/L of tryptophan (where appropriate) and 1 g/L of methionine were used, unless stated otherwise.

Example 3: Development of an Alternative Psilocybin Pathway [0077] After verifying the expected TrpM activity in the bacterial system, the possibility of using TrpM as part of an alternative psilocybin biosynthetic pathway, characterized by early methylation, followed by decarboxylation and phosphorylation, was evaluated (Figure 3). This is in contrast to the previously established psilocybin biosynthesis pathway utilizing norbaeocystin methyltransferase, PsiM, which shows strong preference for phosphorylated substrate, resulting in a terminating methylation step in the biosynthesis. To demonstrate the feasibility of this alternative pathway, each step was evaluated independently.

Example 4: TrpM activity on 4-hvdroxytryptophan

[0078] In the present study, the ability of E. coifs endogenous tryptophan synthase (TrpB) to condense externally supplemented 4-hydroxyindole and serine to efficiently form the nonnatural amino acid, 4-hydroxytryptophan is coupled with TrpM to study the activity of TrpM towards 4-hydroxytryptophan in vivo. Since TrpM activity has been shown to be optimal at 25 °C (Figure 2) and endogenous TrpB in vivo activity in the context of psilocybin biosynthesis has been shown to be optimal at 37°C, a range of relevant temperatures (25, 30, 37, and 42°C) were screened to evaluate the effect of fermentation temperature on joint TrpB- TrpM activity.

[0079] The addition of 4-hydroxyindole at a concentration of 50mg/L was tested with the pETM6-SDM2x-TrpM strain and resulted in production of mono- and di-methylated 4- hydroxytryptophan, but interestingly no tri-methylated 4-hydroxytryptophan was observed (Figure 4). The production of these two compounds was further verified by high resolution LCMS-MS analysis with both MSI and MS2 ions matching with predicted values (Figure 8). This represents the first report of the biosynthesis of methylated 4-hydroxytryptophan and is a key step towards the realization of the alternative psilocybin production pathway presented herein. Results showed a clear optimal temperature of 30°C for the combined TrpB-TrpM biosynthetic pathway and demonstrates the tradeoff between TrpB (optimum at 37°C) and TrpM (optimum at 25 °C).

Example 5: Conversion of methylated tryptophan to methylated tryptamine by PsiD [0080] The next step in the alternative psilocybin pathway is the decarboxylation of methylated 4-hydroxytryptophan, resulting in methylated 4-hydroxytryptamine. Without wishing to be bound by theory, this reaction is hypothesized to be catalyzed by the decarboxylase, PsiD. In native mushrooms, PsiD catalyzes the decarboxylation of tryptophan to tryptamine as the first concerted step of psilocybin biosynthesis. PsiD shows significant substrate flexibility, also converting 4-hydroxytryptophan to 4-hydroxytryptamine at near stoichiometric yield.

[0081] The effect of /V-methylations on PsiD activity was determined. Due to limited commercial availability of /V-methylated 4-hydroxytryptophan, this step of the alternative pathway was first evaluated against /V-methylated tryptophan derived from a chemical methylation.

[0082] To probe the impact of amine group methylations on the activity of PsiD, a chemical methylation tryptophan using iodomethane was performed (Ralph N. Salvatore et al., 1999). This reaction was performed under conditions that would result in a mixture of mono-, di-, and tri-methylated tryptophan. The LCMS analysis of methylated products showed significant quantities of products with m/z of 219, 233, and 247, with retention times that would be expected for /V-methyltryptophan, /V/V-dimethyltryptophan, and JV,JV,JV- trimethyltryptophan, respectively. These reaction products were then semi-purified using preparative HPLC (Figure 5). Due to poor separation between methylated tryptophan products, individually purified products were not possible to be obtained. The product peaks were split into 3 mixed fractions, with each fraction having a different product composition as presented in Table 1.

Table 1. Percent composition of chemically synthesized /V-methylated tryptophan substrate fractions

[0083] These fractions were then concentrated with a rotary evaporator and lyophilized to obtain a solid product that could be supplemented into the fermentation media containing E. coli expressing PsiD. These fractions were made into a stock solution and fed at a concentration of 50 mg/L to BL21 star™ (DE3) containing pETM6-SDM2x-PsiD. The experiment showed the production of /V-methyltryptamine (NMT) in fraction 2 and 3 and /V/V-dimethyltryptamine (DMT) in fractions 3 and 4 (Figure 7). No evidence ofN,N,N- trimethyltryptamine (TMT) was observed, indicating either the steric hindrance of the quaternary amine, that the positively charged side chain was sufficient to block decarboxylation activity, or is indicative of mass transport issues across the cell membrane.

Each of these fractions were tested at 25, 30, 37, and 42°C.

[0084] Once PsiD had been demonstrated to show activity towards both N-methyl- and 4- hydroxy- functionalized tryptophan individually, the conversion of mono- and di-methyl-4- hydroxytryptophan to their corresponding tryptamine components, N-methyl-4- hydroxytryptamine and psilocin, respectively, was sought to be demonstrated. Due to the required disubstituted tryptophans not being commercially available and the low concentrations achieved in the TrpM evaluation study limiting downstream purification, this activity was studied through coupled analysis with TrpB and TrpM in vivo. A co-culture system with one strain capable of exogenous expression of PsiD and another strain capable of endogenous TrpM expression was tested in well plate, shake flask, and 2-L bioreactor platforms at various strain ratios. Upon supplementation with 4-hydroxyindole no production of the desired products was observed. Ongoing studies to identify alternative enzymes capable of performing the desired biotransformation are underway, including evaluation of PsiD homologs from other psilocybin producing organisms (e.g., Psilocybe cubensis, Gymnopilus dilepis, Pcmcieolus cyanescens, and Psilocybe cyanescens) and other decarboxylases such as tryptophan decarboxylase (CrTDC) from Catharanlhus mseus (Milne et al., 2020) and Rgn TDC from Ruminococcus gnavus (McDonald et ai., 2019).

Alternatively, the P450, PsiH, is also being evaluated for its ability to hydroxylate methylated tryptamine products of PsiD. Nevertheless, this reported activity towards mono- and dimethylated tryptophan represents activity towards a new substrate not previously reported for PsiD.

Example 6: AUV-dimethyl-4-hvdroxytrvDtamine (psilocin) into psilocybin

[0085] The final step in this alternative pathway is the phosphorylation of psilocin into psilocybin by means of the 4-hydroxytryptamine kinase, PsiK. In both the natural and recombinant psilocybin biosynthetic pathways PsiK serves a primary function of phosphorylating 4-hydroxytryptamine into norbaeocystin, enabling two sequential methylations by PsiM ultimately resulting in psilocybin. Additionally, PsiK has been shown to play a secondary housekeeping role with activity towards psilocin resulting in the conversion back to the prodrug, psilocybin. Psilocin is only present in natural systems due to spontaneous dephosphorylation or phosphatase activity on psilocybin (Lenz et al., 2020). The body of literature supporting the ability of PsiK to have the requisite activity is well established (Fricke et al., 2020), however, this activity has not been explicitly shown in an in vivo E. coli system to date.

[0086] To confirm the ability of PsiK to convert psilocin into psilocybin in vivo, we first needed to obtain a sample of psilocin to supplement into the fermentation media of our E. coli strain containing plasmid, pETM6-SDM2x-psiK. Psilocin is relatively unstable for long periods of time, so we were unable to store samples on hand. The commercial enzyme, alkaline phosphatase (AP), was utilized to produce psilocin in vitro, which would then be fed to our E. coli strain. First, we needed to establish a heat denaturation protocol to capable of abolishing AP activity, such that AP will no longer be active once supplemented into the fermentation media, enabling psilocybin accumulation.

[0087] To establish this protocol a short experiment was performed where AP lOx buffer was combined with a psilocybin solution in water at a final concentration just over 1 g/L. This solution was incubated at 37°C for 30 minutes with and without enzyme. Following the 30- minute incubation period, all available psilocybin was converted to psilocin (if AP was present) and half of the samples were subjected to heat denaturation at 80°C for 20 minutes, while the other samples were stored at room temperature. To confirm that the denaturation had worked, some psilocybin was supplemented back into all samples. The samples were then placed back into 37°C for 30 minutes. Aliquots were taken periodically throughout the process to determine the psilocybin and psilocin content.

[0088] The samples containing AP that was subjected to heat denaturation showed all the psilocybin added before the heat denaturation was converted to psilocin, and the psilocybin that was added after heat exposure was unchanged (Figure 9C). Additionally, the presence of psilocin made prior to heat denaturation suggests psilocin was able to withstand the heat denaturation process with only minor degradation. The samples containing AP that was not subjected to heat denaturation showed complete conversion of all of the second psilocybin addition to psilocin (Figure 9B). Finally, the no-enzyme controls confirmed the stability of psilocybin in solution with no spontaneous degradation of psilocybin into psilocin detected (Figure 9A).

[0089] Next, we repeated the same alkaline phosphatase reaction and heat denaturation process described above to produce psilocin for supplementation into the culture media. This reaction was supplemented into an E. coli culture expressing PsiK, at a final concentration of 50 mg/L of psilocin about 1 hour post induction of PsiK expression. The culture was incubated at 37 °C for 24 hours, and then subjected to HPLC/LCMS analysis. The results showed a near complete conversion of supplemented psilocin into psilocybin (Figure 10), confirming both the required activity for the proposed pathway and alleviating any concerns regarding transport of psilocin across the cell membrane.

[0090] Efforts towards the production of psilocybin through an alternate biosynthesis pathway are described herein, characterized by initial precursor methylation with TrpM, followed by decarboxylation and phosphorylation. Through evaluation of the proposed pathway feasibility, substrate flexibility of TrpM and PsiD has been further characterized, demonstrating activity towards 4-hydroxytryptophan and mono- and di-methylated tryptophans, respectively. Furthermore, this represents the first report of mono- and di-methyl 4-hydroxytryptophan biosynthesis. Although psilocybin production through this pathway has not yet been achieved, all individual steps have been validated with the exception of the decarboxylation of N,N-dimethyltryptophan. Efforts are underway to validate this step in vivo and demonstrate the first proof-of-principle of this alternative psilocybin biosynthesis approach utilizing TrpM.

Bibliography

[0091] Adams, A.M., Kaplan, N.A., Wei, Z., Brinton, J.D., Monnier, C.S., Enacopol, A.L., Ramelot, T.A., Jones, J.A., 2019. In vivo production of psilocybin in E. coli. Metab. Eng. 56, 111-119. doi: 10. 1016/J.YMBEN.2019.09.009

[0092] Bao, S.H., Jiang, H., Zhu, L.Y., Yao, G., Han, P.G., Wan, X.K., Wang, K., Song, T.Y., Liu, C.J., Wang, S., Zhang, Z.Y., Zhang, D.Y., Meng, E., 2021. A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA. Cell Rep. 36, 109413. doi: 10.1016/J.CELREP.2021.109413

[0093] Blei, F., Fricke, J., Wick, J., Slot, J.C., Hoffmeister, D., 2018. Iterative 1-Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication. ChemBioChem 19, 2160- 2166. doi: 10.1002/CBIC.201800336

[0094] Fricke, J., Blei, F., Hoffmeister, D., 2017. Enzymatic Synthesis of Psilocybin. Angew.

Chemie Int. Ed. 56, 12352-12355. doi: 10.1002/anie.201705489 [0095] Fricke, J., Kargbo, D.R., Regestein, D.L., Lenz, C., Peschel, D.G., Rosenbaum, P.D.M.A., Sherwood, D.A., Hoffmeister, P.D.D., 2020. Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin. Chemistry 26, 8281. doi : 10.1002/CHEM.202000134

[0096] Fricke, J., Sherwood, A., Kargbo, R., Orry, A., Blei, F., Naschberger, A., Rupp, B., Hoffmeister, D., 2019. Enzymatic Route toward 6-Methylated Baeocystin and Psilocybin. ChemBioChem 20, 2824-2829. doi: 10.1002/cbic.201900358

[0097] He, W., Fu, L., Li, G., Andrew Jones, J., Linhardt, R.J., Koffas, M., 2015. Production of chondroitin in metabolically engineered E. coli. Metab. Eng. 27. doi : 10.1016/j .ymben.2014.11.003

[0098] Hoefgen, S., Lin, J., Fricke, J., Stroe, M.C., Mattern, D.J., Kufs, J.E., Hortschansky, P., Brakhage, A.A., Hoffmeister, D., Valiante, V., 2018. Facile assembly and fluorescencebased screening method for heterologous expression of biosynthetic pathways in fungi.

Metab. Eng. 48, 44-51. doi: 10.1016/J.YMBEN.2018.05.014

[0099] Jr., W.J.G., McKinney, M.G., O’Dell, P.J., Bollinger, B.A., Jones, J.A., 2021. Homebrewed Psilocybin: Can New Routes for Pharmaceutical Psilocybin Production Enable Recreational Use? doi: 10.1080/21655979.2021.1987090

[0100] Lenz, C., Wick, J., Braga, D., Garcia-Altares, M., Lackner, G., Hertweck, C., Gressler, M., Hoffmeister, D., 2020. Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms. Angew. Chemie 132, 1466-1470. doi: 10.1002/ANGE.201910175

[0101] Milne, N., Thomsen, P., Molgaard Knudsen, N., Rubaszka, P., Kristensen, M., Borodina, I., 2020. Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives. Metab. Eng. 60, 25-36. doi: 10.1016/j.ymben.2019.12.007

[0102] Ralph N. Salvatore, Advait S. Nagle, Shaun E. Schmidt, and, Jung, K.W., 1999. Cesium Hydroxide Promoted Chemoselective N-Alkylation for the Generally Efficient Synthesis of Secondary Amines. Org. Lett. 1, 1893-1896. doi: 10.1021/OL9910417

[0103] Sherwood, A.M., Halberstadt, A.L., Klein, A.K., McCorvy, J.D., Kaylo, K.W., Kargbo, R.B., Meisenheimer, P., 2020. Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin. J. Nat. Prod, acs.jnatprod.9b01061. doi: 10.1021/acs.jnatprod.9b01061

Table 2. Plasmid and Strain List

Table 3: Sequences

[0104] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.

Claims

CLAIMS What is claimed is:

1. A method for the production of psilocybin or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of TrpM, psiD, psiK, Tdc, and combinations thereof; and culturing the host cell.

2. The method of claim 1, wherein the TrpM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

3. The method of claim 1, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

4. The method of claim 1, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

5. The method of claim 1, wherein the Tdc gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 19, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

6. The method of claim 1, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.

7. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene all under control of a single promoter in operon configuration.

8. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene all under control of a single promoter in operon configuration.

9. The method of claim 7 or 8, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

10. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

11. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

12. The method of claim 10 or 11, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

13. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

14. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

15. The method of claim 13 or 14, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

16. The method of claim 1, wherein the intermediate or side product of psilocybin is 4- hydroxytryptophan, 4-hydroxy-A-methyltryptophan, 4-hydroxy-A, A-dimethyltryptophan, 4- hydroxy-TV, N, A-trimethyltryptophan, 4-hydroxy-A-methyltryptamine (norpsilocin), 4-hydroxy- /V,/V-dimethyltryptamine (psilocin), 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT), N- methyltryptamine (NMT), N, A-dimethyltryptamine (DMT), or N, A, A-trimethyltryptamine (TMT).

17. The method of claim 1, wherein the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4-hydroxytryptophan, 4-hydroxytryptamine, tryptophan, tryptamine, N-methyltryptophan, N,N-dimethyltryptophan and combinations thereof.

18. The method of claim 17, wherein the supplement is fed continuously to the host cell.

19. The method of claim 1, wherein the host cell is grown in an actively growing culture.

20. A recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of TrpM, psiD, psiK, Tdc, and combinations thereof.

21. The recombinant prokaryotic cell of claim 20, wherein the TrpM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto

22. The recombinant prokaryotic cell of claim 20, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

23. The recombinant prokaryotic cell of claim 20, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

24. The recombinant prokaryotic cell of claim 20, wherein the Tdc gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 19, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

25. The recombinant prokaryotic cell of claim 20, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.

26. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a TrpM gene, a psiD gene, and a psiK gene, all under control of a single promoter in operon configuration.

27. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a TrpM gene, a Tdc gene, and a psiK gene, all under control of a single promoter in operon configuration.

28. The recombinant prokaryotic cell of claim 26 or 27, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

29. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

30. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

31. The recombinant prokaryotic cell of claim 29 or 30, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

32. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

33. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

34. The recombinant prokaryotic cell of claim 32 or 33, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

35. An expression vector comprising a TrpM gene, a psiD gene, and a psiK gene all under control of a single promoter in operon configuration.

36. An expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene all under control of a single promoter in operon configuration.

37. The expression vector of claim 35 or 36, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

38. An expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

39. An expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

40. The expression vector of claim 38 or 39, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

41. An expression vector comprising a TrpM gene, a psiD gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

42. An expression vector comprising a TrpM gene, a Tdc gene, and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

43. The expression vector of claim 41 or 42, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

44. A transfection kit comprising the expression vector of any one of claim 35-43.