Biosynthesis of tryptamines and related compounds

Abstract

Provided are methods, prokaryotic host cells, expression vectors, and kits for the production of psilocybin derivatives produced using at least one psilocybin production gene. The methods enable production of psilocybin derivatives and related compounds including non-naturally occurring species. In certain embodiments, a prokaryotic host cell is selected from the group consisting of 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

BIOSYNTHESIS OF TRYPTAMINES AND RELATED COMPOUNDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The instant application claims priority to and the benefit of U.S. Provisional Application No. 63 / 595,961 filed November 3, 2023, the content of which is hereby incorporated by reference in its entirety.FIELD

[0002] The general inventive concepts relate to the field of medical therapeutics and more particularly to improved methods for the biosynthetic production of novel tryptamine drug candidates through enzyme optimization.SEQUENCE LISTING

[0003] The contents of the electronic sequence listing named 27876_04086_SL.xml created on October 25, 2024 , which is 64 KB in size is herein incorporated by reference in its entirety.BACKGROUND

[0004] In the U.S. alone, nearly one in five individuals suffer from a mental health disorder which costs the United States over 200 billion dollars annually (Carhart-Harris et al., 2016; NIMH, 2022). Unfortunately, there has been a rise in these numbers in the recent years as a result of the COVID-19 pandemic (Yarrington et al., 2021), increased social media usage (Taylor et al., 2020), and growing social, financial, and global unrest (Avcin et al., 2011). Currently, many interventions including medications and psychotherapy have proven to be beneficial for many patients, however, there is still a significant population with unmet mental healthcare needs who could benefit from alternative treatment approaches (Carhart-Harris et al., 2016; Gaynes, 2009). These ongoing issues with mental health treatments can be addressed with psychedelic medicine which is a re-emerging field after many psychedelic compounds were deemed to be abused drugs with no medicinal use by the Controlled Substance Act of 1970 (Tupper et al., 2015). The classification of psychedelic substances as Schedule I drugs terminated nearly all active research on these compounds, slowing the development of psychedelic-based treatments for mental health disorders for decades. Recently, clinical research using classical psychedelics has resumed, with early results implementing controlled, therapist mediated sessions with participating patients (Tupper et al., 2015) to study its therapeutic efficacy for post-traumatic stress disorder (PTSD) (Gluff et al., 2017; Johnson and Griffiths, 2017), depression (Carhart-Harris et al., 2017, 2016), obsessive compulsive disorder(OCD) (Matsushima et al., 2009), and addiction (Garcia-Romeu et al., 2014). Compounds such as 4-phosphoryloxy-7V,7V-dimethyltryptamine (psilocybin), the active and “naturally occurring” component in ‘magic’ mushrooms, is an up and coming therapeutic drug candidate for these clinical trials as results have indicated reduced depressive episodes for months at a time, often after only one or two treatments (Ziff et al., 2022). Due to the promising results of these ongoing clinical trials, there are interests in non-natural psilocybin derivatives as potential new drug candidates (Flower et al., 2023).

[0005] Psilocybin has also been shown to result in fewer occurrences of serious adverse effects compared to other psychedelic compounds (de Veen et al., 2017; MacCallum et al., 2022). Psilocybin is a prodrug, which is rapidly dephosphorylated in vivo, forming psilocin, which binds a range of serotonin receptors due to its structural similarity to serotonin, a neurotransmitter that is responsible for controlling mood (Cao et al., 2022)(Dinis-Oliveira, 2017). Specifically, binding at the 5-HT2A receptor is thought to give rise to the hallucinogenic experiences which are a hallmark of classical psychedelics.

[0006] Psilocybin treatment is, however, not perfect. While it has been shown to be safe and non-addictive, there is one significant side effect that cannot be ignored, a roughly 6-hour hallucinogenic experience which begins approximately 20-40 minutes after consuming the drug (MacCallum et al., 2022). Neuroscientists are beginning to unravel the mechanism of mental health treatment by psychedelics and have suggested that the presence of a hallucinogenic experience may not be necessary to achieve positive mental health impacts (Cameron et al., 2021). Thus a need exists for a larger drug candidate pool with diverse efficacies and psychedelic properties.SUMMARY

[0007] The general inventive concepts relate to and contemplate methods and compositions for producing novel tryptamines and psilocybin analogs (also referred to herein as derivatives) via biosynthesis. The general inventive concepts are based on the discovery that certain substituted indole or tryptophan materials are not viable substrates for the traditional psilocybin biosynthetic pathway. Applicants have discovered novel combinations of enzymes and cocultures that provide a viable pathway for the production of novel tryptamines and psilocybin analogs that could not be produced according to conventional methods or are only produced in minor amounts.

[0008] The general inventive concepts relate to the use of promiscuous enzyme activity to enable and enhance the production of tryptamine derivatives and psilocybin derivatives with the goal of producing a variety of pharmaceutical candidate compounds for treatment of an array of disorders. Specifically, the examples provided herein detail the expansion of biosynthetic capabilities through feeding of derivatized indole substrates as well as the use of diverse tryptophan decarboxylases, tryptophan synthases, halogenase, and prenyltransferase enzymes together with previously established psilocybin biosynthetic genes (e.g., PsiH, PsiK, and PsiM) for the production of diverse psilocybin derivative compounds.

[0009] The general inventive concepts also contemplate the novel combination of cell cultures comprising enzymes with activity that produce psilocybins and related compounds (e.g., conventional enzymes such as psiK, psiD, and psiM, among others) with “tryptophan modifying enzymes” i.e., those having activity that modifies naturally occurring tryptophans to produce new and novel tryptophans, tryptamines, and psilocybins that otherwise were not previously achievable via biosynthesis (also referred to herein as non-naturally occurring).

[0010] In addition, the general inventive concepts recognize that certain of the traditional enzymes lack activity on specific substrates (e.g., some indole derivatives, tryptophan derivatives, and tryptamine derivatives and other related pathway intermediates). Thus, the general inventive concepts are also based on the discovery that certain conventional psilocybin producing enzymes (e.g., psiD) can be replaced or substituted in the biosynthetic pathway with an enzyme having the same general activity (e.g., decarboxylase) but has a varied scope of substrates on which it will act.

[0011] Provided is a method for the production of a non-naturally occurring tryptophan, a non- naturally occurring tryptamine, or a non-naturally occurring psilocybin, comprising administering a substrate to a cell culture, the cell culture comprising a recombinant prokaryotic cell having enzymatic activity selected from hydroxylase activity, decarboxylase activity, kinase activity, A-m ethyl transferase activity, and a prokaryotic cell having enzymatic activity selected from tryptophan synthase activity, halogenase activity, prenyltransferase activity, and combinations thereof.

[0012] Also provided is an expression vector comprising at least one gene selected from PsiH, CPR, RgnTDC, CroTDC, PsmH, PsiD, PsiK, Tri, Q90, Azul, PyrH, SttH, ThHal, RebF, PsiM, 5DMATS, PriB, and 7DMATS and combinations thereof. In certain embodiments, each geneis under control of a separate promoter in monocistronic configuration, optionally wherein each promoter is independently selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, LacUV5, tac, trc, GAP, and xylA promoter.

[0013] Also provided is a culture comprising a cell strain selected from: a first recombinant prokaryotic cell comprising an enzyme having hydroxylase activity, said enzyme being selected from one or both of PsiH and CPR; a second recombinant prokaryotic cell comprising an enzyme having decarboxylase activity, said enzyme being selected from RgnTDC, CroTDC, PsmH, PsiD, and combinations thereof; a third recombinant prokaryotic cell comprising an enzyme having kinase activity, said kinase activity being supplied by PsiK; a fourth recombinant prokaryotic cell comprising an enzyme having tryptophan synthase activity, said enzyme being selected from Tri, Q90 and Azul, and combinations thereof; a fifth recombinant prokaryotic cell comprising an enzyme having halogenase activity, said enzyme being selected from PyrH, SttH, ThHal, RebF, and combinations thereof; a sixth recombinant prokaryotic cell comprising an enzyme having A-m ethyl transferase activity, said N-m ethyl transferase activity being supplied by PsiM; and a seventh recombinant prokaryotic cell comprising an enzyme having prenyltransferase activity, said prenyltransferase activity being supplied by 5DMATS, PriB, 7DMATS, and combinations thereof.

[0014] Also provided is a co-culture comprising at least two cell strains, said at least two cell strains, collectively having enzymatic activity of at least two, at least three, at least four, at least five, at least six, or all seven of the following: hydroxylase activity; decarboxylase activity; kinase activity; tryptophan synthase activity; halogenase activity; prenyltransferase activity; and A -methyl transferase activity.

[0015] Also provided is a recombinant prokaryotic cell comprising one or more expression vector comprising a gene selected from of PsiH, CPR, RgnTDC, CroTDC, PsmH, PsiD, PsiK, Tri, Q90, Azul, PyrH, SttH, ThHal, RebF, PsiM, 5DMATS, PriB, 7DMATS and combinations thereof. In certain embodiments, the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli. Corynebacterium glutamicum, Vibrio natriegens, Bacillus sublilis. Bacillus megalerium. Escherichia coli Nissle 1917, Clostridium acelobullyicum. Streptomyces coelicolor. Lactococcus laclis. Pseudomonas putida, Streptomyces clavuligerus. and Streptomyces venezuelae .

[0016] Also provided is a composition comprising 7-cyanopsilocybin, having the structure:

[0017] Also provided is a composition comprising 7-ethylpsilocybin, having the structure:

[0018] Also provided is a composition comprising 7-carboxaldehy depsilocybin, having the structure:BRIEF DESCRIPTION OF THE FIGURES

[0019] Fig. 1 A is a LCMS graph showing 6-fluoropsilocybin production in a PsiH:DKM coculture (+) control and through feeding of 6-fluoro-4-hydroxyindole.

[0020] Fig. IB is a mass spectrum of the psilocybin derivative of Figure 1 A indicating a m / z 303.

[0021] Fig. 1C is a LCMS graph showing 6-methylpsilocybin production in a PsiH:DKM coculture (+) control and through feeding of 6-methyl-4-hydroxyindole.

[0022] Fig. ID is a mass spectrum of the psilocybin derivative of Figure 1C indicating a m / z 299.

[0023] Fig. IE is a LCMS graph showing 7-chloropsilocybin production in a PsiH:DKM coculture (+) control and through feeding 7-chloro-4-hydroxyindole.

[0024] Fig. IF is a mass spectrum of the psilocybin derivative of Figure IE indicating a m / z 319.

[0025] Fig. 2A is a bar graph showing derivative psilocybin production with supplemented double-substituted indoles made with strains pSilol6 and Gymdi30 with 50 mg / L disubstituted indole supplement.

[0026] Fig. 2B is a graph showing peak areas for psilocybin derivatives produced by pSilol6 with supplementation of 50 mg / L single-substituted, di substituted, 4-hydroxyindole, or no indole. Error bars signify standard error of the mean for replicates (N=2). indicates statistical significance (p<0.05).

[0027] Fig. 3 A is a bar graph showing double-substituted indoles remaining from fermentations containing pSilol6 and Gymdi30 psilocybin production strains.

[0028] Fig. 3B is a graph showing the presence of double-substituted indoles in AMM measured at 0 hr, 24 hr, and 48 hr time points. Pairwise comparisons found no statistical significance within each indole group. Error bars signify standard error of the mean for replicates (N=2).

[0029] Fig. 4A is a graph showing toxicity evaluation of double-substituted indoles supplemented to psilocybin production strains pSilol6 and Gymdi30 at 25 mg / L, 50 mg / L, and 100 mg / L of 7Cl,4OH-indole.

[0030] Fig. 4B is a graph showing toxicity evaluation of double-substituted indoles supplemented to psilocybin production strains pSilol6 and Gymdi30 at 25 mg / L, 50 mg / L, and 100 mg / L of 6Me,4OH-indole.

[0031] Fig. 4C is a graph showing toxicity evaluation of double-substituted indoles supplemented to psilocybin production strains pSilol6 and Gymdi30 at 25 mg / L, 50 mg / L, and 100 mg / L of 6F,4OH-indole.

[0032] Fig. 5 is a graph showing the results of RgnTDC activity against a range of tryptophan derivative substrates.

[0033] Fig. 6 is a graph showing the results of CroTDC activity against a range of tryptophan derivative substrates.

[0034] Fig. 7 is a graph showing the results of PsmH activity against a range of tryptophan derivative substrates.

[0035] Fig. 8 is a graph showing measured activity of diverse decarboxylases on the natural amino acid tryptophan. Tryptophan provided at 1 g / L in AMM media at 37 °C. PsiD (green) and RgnTDC (red) shown tryptamine production, while PsmH (yellow) and empty vector negative control (blue) shows no tryptamine production.

[0036] Fig. 9 shows the mass spectroscopy extracted ion chromatographs of 7- cyanopsilocybin production is shown in blue with the no indole control shown in black. The structure of 7-cyanopsilocybin is shown with a retention time of approximately 4.0 min.

[0037] Fig. 10 shows the mass spectroscopy extracted ion chromatographs of 7- ethylpsilocybin production is shown in blue with the no indole control shown in black. The structure of 7-ethylpsilocybin is shown with a retention time of approximately 5.5 min.

[0038] Fig. 11 shows the mass spectroscopy extracted ion chromatographs of 7- carboxaldehydepsilocybin production is shown in blue with the no indole control shown in black. The structure of 7-carboxaldehydepsilocybin is shown with a retention time of approximately 3.8 min.

[0039] Fig. 12 is a table showing the yield of tryptophan synthesis from indole derivatives for Q90 tryptophan synthase.

[0040] Fig. 13 is a table showing the yield of tryptophan synthesis from indole derivatives for Tri tryptophan synthase.

[0041] Fig. 14 is a table showing they yield of tryptophan synthesis from indole derivatives for Azul tryptophan synthase.

[0042] Fig. 15 is a table showing a summary of tryptamine production from co-culture expressing Q90 tryptamine synthase and RgnTDC.

[0043] Fig. 16A is a graph showing chlorotryptophan production by halogenases in a LCMS extracted ion chromatograph (EIC) shown in pink, Q90 positive control supplemented with chloroindole in black, and empty vector negative control in gray.

[0044] Fig. 16B is a mass spectrum of chlorotryptophan indicating a m / z 239.

[0045] Fig. 16C is a graph showing remaining tryptophan and chlorotryptophan production by each halogenase (PyrH, SttH, ThHal, and RebH) with and without flavin reductase, RebF. The tryptophan synthase (Q90) positive control is supplemented with 7-chloroindole. Error bars signify standard error of the mean for replicates (N=3). Reaction products above limit of detection by LCMS, but below limit of quantification by HPLC.

[0046] Fig. 17A is a graph showing chloro,4-hydoxytryptophan production by halogenases via product specific LCMS extracted ion chromatograph shown in pink, TrpB positive control supplemented with chloro-4-hydroxyindole in black, and empty vector negative control in gray. All EICs were taken under a m / z 255.

[0047] Fig. 17B is a mass spectrum of chlorotryptophan indicating a m / z 255.

[0048] Fig. 17C is a graph showing chlorinated 4-hydroxytryptophan production by each halogenase, with and without the reductase partner. The tryptophan synthase (TrpB) positive control is supplemented with 7-chloro-4-hydroxyindole. Error bars signify standard error of the mean for replicates (N=3). Reaction products above limit of detection by LCMS, but below limit of quantification by HPLC.

[0049] Fig. 18 is a graph showing bromination of tryptophan by various halogenases under various concentrations of bromide with 1 g / L tryptophan media supplement.

[0050] Fig. 19 is a graph showing the extracted ion chromatograms for 4-hydroxy- dimethylallyltrytophan production from a supplement of 4-hydroxyindole in A. coli BL21 star™ (DE3). The asterisk (*) above indicates the product peak at around 7.5 minutes.DETAILED DESCRIPTION

[0051] 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.

[0052] 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.

[0053] 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.

[0054] “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.

[0055] 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.

[0056] 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. All ranges and parameters, including but not limited to percentages, parts, and ratios, disclosed herein are understood to encompass any and all subranges assumed and subsumed therein, and every number between the endpoints. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more (e.g., 1 to 6.1), and ending with a maximum value of 10 or less (e.g., 2.3 to 9.4, 3 to 8, 4 to 7), and finally to each number 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 contained within the range.

[0057] As used herein, the term “prokaryotic 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.

[0058] As used herein, the term “recombinant” when referring to cells or host cells 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 geneticmodification comprises integrating the polynucleotide in the genome of the host cell. In further embodiments the polynucleotide is exogenous in the host cell.

[0059] As used herein, the term “non-naturally occurring” when used to describe or refer to a tryptophan derivative, tryptamine derivative, or psilocybin derivative, refers to molecular species or substrate that are not fermented by (when referring to e.g., indoles and tryptophans) or produced by the conventional psilocybin biosynthetic pathway. Tryptophan, tryptamine, 4- hydroxytryptamine, norbaeocystin, baeocystin, and psilocybin are considered naturally occurring substrates and products. In contrast, derivatives of the naturally occurring substrates (e.g., 7-cyanoindole) or substituted versions of psilocybins (e.g., 2-chloropsilocybin) are non- naturally occurring. In certain embodiments, “non-naturally occurring” refers to products resulting from fermentation of substrates that show little to no decarboxylation via psiD. In certain embodiments, the non-naturally occurring compound refers to specific substrate compounds such as a 2-chloro, 2-methyl, 6-cyano, 7-cyano, 7-iodo, 7-methoxy, and 7-nitro derivative of indole and / or tryptophan.

[0060] As used herein, the term “intermediate” means an intermediate in the production or biosynthesis of a tryptophan or a tryptamine. For example, an intermediate of psilocybin means, e.g., norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine.

[0061] As used herein, the term “side product” of a tryptophan or a tryptamine means a side product in the production or biosynthesis of a tryptophan or a tryptamine. For example, a side product of psilocybin means, e.g., aeruginascin, psilocin, norpsilocin, or 4-hydroxy-A,A,A- trimethyltryptamine (4-OH-TMT).

[0062] As used herein, the term “derivative” of a compound means a compound with one or more side chain additions or subtractions that still maintains the chemical classification of the compound. For example, a derivative of a tryptophan or a tryptamine means a tryptophan or a tryptamine with one or more side chain functional additions or subtractions that still maintains the chemical classification of the tryptophan or tryptamine. In some embodiments, the derivative is non-naturally occurring. In some embodiments, the derivative includes an addition or functional group at one or more of the 1, 2, 3, 4, 5, 6, or 7 position of the indole ring numbering classification. In certain embodiments, the derivative refers to a 2-chloro, 2-methyl, 6-cyano, 7-cyano, 7-iodo, 7-methoxy, and 7-nitro derivative.

[0063] As previously mentioned, the general inventive concepts relate to the use of promiscuous enzyme activity to enable and enhance the production of tryptamine derivatives (including non-naturally occurring derivatives) with the goal of producing a variety of pharmaceutical candidate compounds for treatment of an array of disorders. Specifically, the examples provided herein detail the expansion of biosynthetic capabilities through feeding of derivatized indole substrates (Example #1) as well as the use of diverse tryptophan decarboxylases (Example #2 and #4), tryptophan synthases (Example #3 and #4), prenyltransferase enzymes (Example #6), and halogenase enzymes (Example #5) (alone or in combination) together with previously established psilocybin biosynthetic genes (PsiH, PsiK, and / or PsiM) for the production of diverse psilocybin derivative compounds. The screening and evaluation of these biosynthetic modules was accomplished using an E. coli host strain with frequent application of cocultures and tripartite cultures (tri-cultures) where 2 or 3 strains, respectively, were grown in the same fermentation vessel to realize proof-of-principle biosynthetic capabilities.

[0064] The materials, compositions, and methods described herein are intended to be used to provide novel routes for the production of non-naturally occurring psilocybins, tryptamines and tryptophans. Previous work has shown methods for using prokaryotic cells to produce psilocybin and related compounds. See e.g., WO2021 / 086513; WO2023 / 081829; and WO 2023 / 081837, the contents of which are hereby incorporated by reference in their entirety.

[0065] Provided is a method for the production of a non-naturally occurring tryptophan, a non- naturally occurring tryptamine, or a non-naturally occurring psilocybin, comprising administering a substrate to a cell culture, the cell culture comprising a recombinant prokaryotic cell having enzymatic activity selected from hydroxylase activity, decarboxylase activity, kinase activity, / ' -methyl transferase activity, and a prokaryotic cell having enzymatic activity selected from tryptophan synthase activity, halogenase activity, prenyltransferase activity, and combinations thereof.

[0066] In certain embodiments, the recombinant cell is a prokaryotic cell. In certain exemplary embodiments, the prokaryotic cell is selected from Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus sublilis. Bacillus megalerium, Escherichia coli Nissle 1917, Clostridium acelobullyicum, Streptomyces coelicolor. Lactococcus laclis. Pseudomonasputida, Streptomyces clavuligerus, and Streptomyces venezuelae. In certain exemplary embodiments, the prokaryotic cell is an A. coli cell.

[0067] In certain embodiments, the enzymatic activity is provided by one or more of the following: RgnTDC, CroTDC, PsmH, Tri, Q90, Azul, PyrH, SttH, ThHal, RebF, PsiH, CPR, PsiD, PsiK, PsiM, 5DMATS, PriB, and 7DMATS. In certain embodiments, the hydroxylase activity is supplied by an enzyme selected from one or both of PsiH and CPR. In certain embodiments, the decarboxylase activity is supplied by an enzyme selected from RgnTDC, CroTDC, PsmH, PsiD, and combinations thereof. In certain embodiments, the kinase activity is supplied by PsiK. In certain embodiments, the tryptophan synthase activity is supplied by an enzyme selected from TrpB, Tri, Q90, Azul, and combinations thereof. In certain embodiments, the halogenase activity is supplied by an enzyme selected from PyrH, SttH, ThHal, RebF, and combinations thereof. In certain embodiments, the A-methyltransferase activity is supplied by PsiM. In certain embodiments, the prenyltransferase activity is supplied by 5DMATS, PriB, 7DMATS, and combinations thereof. In certain embodiments, the enzymatic activity is at least two of, at least three of, at least four of, at least five, at least six, and at least seven of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthase activity, halogenase activity, A-methyltransferase activity, and prenyltransferase activity. In certain embodiments, the enzymatic activity is each of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthase activity, halogenase activity, N- methyltransferase activity, and prenyltransferase activity.

[0068] In certain embodiments, the PyrH gene is encoded by a nucleotide sequence comprising SEQ ID NO: 12, 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 PyrH gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, 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.

[0069] In certain embodiments, the SttH gene is encoded by a nucleotide sequence comprising SEQ ID NO: 14, 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 SttH gene encodes a polypeptide comprising theamino acid sequence of SEQ ID NO: 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.

[0070] In certain embodiments, the ThHal gene is encoded by a nucleotide sequence comprising 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 ThHal gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 17, 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.

[0071] In certain embodiments, the RebF gene is encoded by a nucleotide sequence comprising SEQ ID NO: 18, 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 RebF gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 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.

[0072] In certain embodiments, the PsmH gene is encoded by a nucleotide sequence comprising SEQ ID NO: 20, 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 PsmH gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 21, 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.

[0073] In certain embodiments, the PsiD gene is encoded by a nucleotide sequence comprising SEQ ID NO: 23, 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 SEQ ID NO: 22, 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.

[0074] In certain embodiments, the PsiK gene is encoded by a nucleotide sequence comprising SEQ ID NO: 25, 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 SEQ ID NO: 24, 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.

[0075] In certain embodiments, the PsiM gene is encoded by a nucleotide sequence comprising SEQ ID NO: 27, 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 PsiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 26, 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.

[0076] In certain embodiments, the RgnTDC gene is encoded by a nucleotide sequence comprising SEQ ID NO: 28, 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 RgnTDC gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 29, 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.

[0077] In certain embodiments, the CroTDC gene is encoded by a nucleotide sequence comprising SEQ ID NO: 30, 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 CroTDC gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31, 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.

[0078] In certain embodiments, the Q90 gene is encoded by a nucleotide sequence comprising SEQ ID NO: 32, 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% sequenceidentity thereto. In certain embodiments, the Q90 gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33, 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.

[0079] In certain embodiments, the Tri gene is encoded by a nucleotide sequence comprising SEQ ID NO: 34, 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 Tri gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 35, 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.

[0080] In certain embodiments, the Azul gene is encoded by a nucleotide sequence comprising SEQ ID NO: 36, 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 Azul gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 37, 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.

[0081] In certain embodiments, the 5DMATS gene is encoded by a nucleotide sequence comprising SEQ ID NO: 38, 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 5DMATS gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 39, 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.

[0082] In certain embodiments, the PriB gene is encoded by a nucleotide sequence comprising SEQ ID NO: 40, 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 PriB gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 41, or a sequence having at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0083] In certain embodiments, the 7DMATS gene is encoded by a nucleotide sequence comprising SEQ ID NO: 42, 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 7DMATS gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 43, 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.

[0084] In certain embodiments, the enzyme or enzymatic activity is supplied by an expression vector supplied to or contacted by the cell. The result is a recombinant prokaryotic cell comprising one or more expression vectors that provide enzymatic activity described herein. More specifically, the expression vector provides one or more of RgnTDC, CroTDC, PsmH, Tri, Q90, Azul, PyrH, SttH, ThHal, RebF, PsiH, CPR, PsiD, PsiK, PsiM, 5DMATS, PriB, and 7DMATS, as described herein. In some embodiments, the cells and vectors comprise a promoter and the promoter is selected from G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.

[0085] It is envisaged that many derivatives of intermediates or side products of psilocybin may be produced by any of the methods described herein. In some embodiments, the derivatized intermediate or side product of psilocybin is a derivatized version of one or more of norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy-A,A,A-trimethyltryptamine (4-OH-TMT). The derivatized version of these compounds may be the result of either culturing a cell with a derivatized substrate of e.g., indole, or of enzymatic activity provided by the expression vectors and recombinant cells (e.g., prenyltransferase) discussed herein, or a combination thereof.

[0086] In certain embodiments, the cell is cultured with a supplement independently selected from the group consisting of indole, a derivatized indole (e.g., 7-cyanoindole), tryptophan, a derivatized tryptophan, tryptamine, and a derivatized tryptamine. The derivatized indoles, tryptophans, and tryptamines generally refer to those species having a functional group selected from acyl halide, aldehyde, alkene, alkyne, amide, carboxamide, amino, azide, boronate, bromo, carboxylic acid, chloro, cyanate, cyano, ester, ethyl, fluoro, hydroxy, iodo, isocyanate,isopropyl, isothiocyanate, methoxy, methyl, nitrate, nitrite, nitro, propyl, sulfinic acid, thiocyanate, thial, thiol, trifluoromethyl, trifluoromethoxy, butyl, and combinations thereof. In certain embodiments, the functional group is present on any of the 1, 2, 3, 4, 5, 6, and 7 positions. In certain embodiments, the functional group is present on any of the 5, 6, and 7 positions. Within this list of potential substrates, the 2-chloro, 2-methyl, 6-cyano, 7-cyano, and 7-nitro derivatives represent substrates not processed by PsiD, while 7-iodo and 7-methoxy are not processed to a suitable degree. In certain embodiments, the culture is mono-culture. In certain embodiments, the culture is a co-culture comprising at least two strains, at least three strains, at least four strains, at least five strains, at least six strains, or at least 7 strains, inclusive.

[0087] In certain embodiments, the general inventive concepts contemplate a co-culture (for producing the compounds discussed herein) comprising a cell strain selected from: a first recombinant prokaryotic cell comprising an enzyme having hydroxylase activity, said enzyme being selected from one or both of PsiH and CPR; a second recombinant prokaryotic cell comprising an enzyme having decarboxylase activity, said enzyme being selected from RgnTDC, CroTDC, PsmH, PsiD, and combinations thereof; a third recombinant prokaryotic cell comprising an enzyme having kinase activity, said kinase activity being supplied by PsiK; a fourth recombinant prokaryotic cell comprising an enzyme having tryptophan synthase activity, said enzyme being selected from Tri, Q90 and Azul, and combinations thereof; a fifth recombinant prokaryotic cell comprising an enzyme having halogenase activity, said enzyme being selected from PyrH, SttH, ThHal, RebF, and combinations thereof; a sixth recombinant prokaryotic cell comprising an enzyme having N-m ethyl transferase activity, said N- methyltransferase activity being supplied by PsiM; and a seventh recombinant prokaryotic cell comprising an enzyme having prenyltransferase activity, said prenyltransferase activity being supplied by 5DMATS, PriB, 7DMATS, and combinations thereof.

[0088] In certain embodiments, the general inventive concepts contemplate a co-culture comprising at least two cell strains, said at least two cell strains, collectively having enzymatic activity of at least two, at least three, at least four, at least five, at least six, or all seven of the following: hydroxylase activity; decarboxylase activity; kinase activity; tryptophan synthase activity; halogenase activity; A-methyltransferase activity, and prenyltransferase activity.

[0089] 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. Continuousfeeding 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.

[0090] In certain embodiments, the resulting non-naturally occurring psilocybins and intermediates 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.Kits

[0091] 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.

[0092] In certain embodiments, the general inventive concepts also provide novel psilocybin derivatives. In certain embodiments, the general inventive concepts provide a composition comprising 7-cyanopsilocybin, having the structure:

[0093] In certain embodiments, the general inventive concepts provide a composition comprising7-ethylpsilocybin, having the structure:

[0094] In certain embodiments, the general inventive concepts provide a composition comprising 7-carboxaldehy depsilocybin, having the structure:EXAMPLES

[0095] The following examples illustrate features and / or advantages of the compositions and methods according to the general inventive concepts. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the general inventive concepts, as many variations thereof are possible without departing from the spirit and scope of the general inventive concepts.Example #1 : Non-Natural psilocybin derivatives starting with disubstituted indoles

[0096] Applicants previously were able to use a co-culture-based approach to successfully feed 13 single substituted indole derivatives to produce 13 non-natural psilocybin derivative compounds (Flower et al., 2023a). The titers achieved using this approach were limited by the low activity of the hydroxylase module (PsiH-CPR) in E. coh. but the proof-of-principal studysuggested a high degree of promiscuity in the remaining pathway enzymes, TrpB, PsiD, PsiK, and PsiM. This conclusion prompts further investigation into feeding our previously optimized psilocybin production strains with disubstituted indole derivatives with one of the substitutions being 4-hydroxy to circumvent limitations of the hydroxylase module. These indoles can range from a 4-hydroxyindole substrate substituted with 5, 6, or 7 position substitutions of a chlorine, methyl, fluorine, bromine, etc.

[0097] The select substrates used in the following example were chosen based on the data gathered from our previous study as well as cost and commercial availability of the required substrates. Due to the successful processing of 6-fluroindole, 6-methylindole, and 7- chloroindole through our production pathway, 6-fluoro-lH-indol-4-ol, 6-methyl-lH-indol-4- ol, and 7-chloro-lH-indol-4-ol will be tested in separate monoculture studies with our top two psilocybin production strains, pSilol6 (Adams et al., 2019) and Gymdi30 (McKinney et al. 2023 MS Thesis Miami University).

[0098] In the published co-culture study carried out with 49 single substituted indoles, 13 of those indoles successfully processed through our psilocybin pathway (Flower et al., 2023a). Specifically, 7-chloroindole produced the highest Extracted Ion Chromatogram (EIC) peak area for the derivative psilocybin amongst all psilocybin derivatives produced at approximately 3.4 x 105(counts*min) with little to no baeocystin intermediate present, which indicates efficient methylation by PsiM. Therefore, we know that this indole can process through the psilocybin pathway genes efficiently. This has led our interest towards the use of disubstituted indole, 7-chloro-lH-indol-4-ol, to result in the production of 7-chloropsilocybin. Another promising candidate for this disubstituted indole approach is 6-methyl-lH-indol-4-ol. While at very low yields, this disubstituted indole was able to process through an in vitro system to produce 6-methylpsilocybin (Fricke, Sherwood, et al., 2019). Our lab was also able to confirm processing of the single substituted indole, 6-methylindole, through our system with an EIC peak area of roughly 1.1 x 105counts*min (Flower et al., 2023a). That being said, LCMS data showed a small peak for baeocystin, which may indicate some biosynthetic limitations upon scaleup as well as a need for HPLC method development to ensure that the two similar compounds have differing retention times. Lastly, we have interest in fluorinated derivatives due to the enhanced pharmacological properties of fluorinated drugs, including enhanced activity, a longer half-life, and overall increased absorption within the tissues (Zhou et al., 2016). Our previous studies indicated that out of all of the 6-position single substituted indoles, 6-fluoroindole produced the highest observed peak area for derivative psilocybin with limitedbaeocystin remaining. Lower quantities of baeocystin could suggest that methylation is not a limiting step, thus allowing for increased production of the derivative psilocybin product.

[0099] These double-substituted indoles were tested using the same process as the single substituted indoles; however, the hydroxylase module will not be included, allowing for testing in monoculture fermentations. The studies address concerns regarding the abilities of the pathway enzymes to use the larger substrates as well as their potential toxicity. Preliminary work supports the ability for the latter pathway enzymes (PsiK and PsiM) to use these substrates, but the ability of TrpB and PsiD to uptake the larger, derivatized substrates and the membrane transport of these compounds was previously unknown.

[0100] Results: The double-substituted indoles were each evaluated using pSilol6 and Gymdi30. We were able to achieve the biosynthesis of three non-natural psilocybin derivatives, including: 6-fluoropsilocybin, 6-methylpsilocybin, and 7-chloropsilocybin (Fig. 1).

[0101] HPLC-MS analysis showed that pSilol6 produced higher EIC peak areas for 7- chloropsilocybin whereas Gymdi30 produced a higher EIC peak area for 6-methylpsilocybin and 6-flouropsilocybin, with the latter not achieving statistical significance (Fig. 2A). The varying trends in these results indicated that pSilol6 may process 7-position substituted indoles more efficiently than Gymdi30, whereas Gymdi30 may be a more suitable strain for processing 6-position indoles and indoles with bulkier side groups. Due to the work that was completed with the single-substituted indoles utilizing pSilol6, subsequent studies on double-substituted indoles also employed pSilol6 for consistency. Nevertheless, it is worth highlighting that Gymdi30 is also capable of processing these doubles substituted indoles, potentially yielding slightly higher derivative psilocybin titers.

[0102] This second approach was hypothesized to outperform our results using singlesubstituted indoles due to the elimination of the PsiH bottleneck, however, derivative psilocybin production using double-substituted indoles was low and, in the case of 7-C1- psilocybin, production was significantly lower than the production using the single-substituted indole (Fig. 2B). This observation could be explained by low substrate or product stability and / or low activity of the pathway enzymes. It was noted that there is a significant amount of indole remaining in each fermentation 24 hours post inoculation (Figure 3A), which suggests substrate stability is likely high enough to be nonlimiting. This was also confirmed by a stability study which measured the concentration of each double-substituted indole in media without cells under fermentation conditions over the course of 48 hours (Fig. 3B). Based onthese conclusions, it is most likely that both the single-substituted and double-substituted indole approaches are significantly limited by our pathway enzymes.

[0103] Building off of this, we explored the toxicity of these compounds through exposure of each psilocybin production strain to a range of concentrations of each disubstituted indole: 25 mg / L, 50 mg / L, and 100 mg / L (Fig. 4). In this work we focused on the impact on growth / final ODeoo (a measure of toxicity). Across the range of concentrations tested, we observed no statistically significant growth deficits for the tested range of indole concentrations (p > 0.05).

[0104] While the biosynthetic pathway has shown that it can process a variety of doublesubstituted indoles, additional preliminary testing was done to evaluate this approach. Unlike the single-substituted indole approach which was determined to be limited by PsiH and requires further optimization outside the scope of this work, evidence suggests that there are accessible opportunities for further improvement of the double- substituted indoles system. Based on our previous findings, it is likely that the double-substituted indoles or intermediate products that are formed from these indoles are pathway limiting, meaning the tryptophan synthase module cannot process these larger substrates efficiently.Example #2: Evaluation of new Decarboxylases enable non-natural psilocybin derivatives

[0105] Previous reports have identified the tryptophan decarboxylase, PsiD, to show significant substrate flexibility, enabling the production of a range of tryptamine derivatives, some of which were able to be converted to psilocybin derivatives (Flower et al. 2023). While proof of principle studies were successful, several notable derivatives, including 2-chloro, 2- methyl, 6-cyano, 6-methoxy, 7-cyano, 7-iodo, 7-methoxy, 7-trifluoromethyl, and 7-nitro demonstrated low or no yield through psiD (Flower et al. 2023). To counter this, we have begun to evaluate RgnTDC (Fig. 4), CroTDC (Fig. 5), and PsmH (Fig. 6) as alternative decarboxylases. We evaluated the promiscuity of these alternative enzymes against the targeted substrates identified above and have successfully observed enhanced activity for all problematic substrates tested using one of the decarboxylases identified above. In all cases, a library of 7 promoters was evaluated for each substrate-enzyme combination. These promoters consisted of two constitutive promoters (XylA and GAP) and 5 inducible T7 variants (G6, H9, H10, C4, and consensus T7). Generally, a high sensitivity to promoter type and strength was observed, especially for higher performing combinations, further illustrating the need for finetuned screening of genetic constructs to achieve optimal performance.

[0106] Promoters that may be used for the instant disclosure include: H10 mutant T7 promoter (taatacgact cactacggaa gaa (SEQ ID NO: 1)); G6 mutant T7 promoter (taatacgact cactatttcg gaa (SEQ ID NO: 2)); H9 mutant T7 promoter (taatacgact cactaatact gaa (SEQ ID NO: 3)); C4 mutant T7 promoter(taatacgact cactatcaag gaa (SEQ ID NO: 4)); consensus T7 promoter (taatacgact cactataggg gaa (SEQ ID NO: 5)); Lac promoter (tttacacttt atgcttccgg ctcgtatgtt g (SEQ ID NO: 6)); Lac UV5 promoter (tttacacttt atgcttccgg ctcgtataat g (SEQ ID NO: 7)); tac promoter (ttgacaatta atcatcggct cgtataatg (SEQ ID NO: 8)); trc promoter (ttgacaatta atcatccggc tcgtataatg (SEQ ID NO: 9)); GAP promoter (gcgtaatgct taggcacagg attgatttgt cgcaatgatt gacacgattc cgcttgacgc tgcgtaaggt ttttgtaatt ttacaggcaa ccttttattc a (SEQ ID NO: 10)); and xylA promoter (ttgaaataaa catttatttt gtatatgatg agataaagtt agtttattgg ataaacaaac taactcaatt aagatagttg atggataaac tt (SEQ ID NO: 11)).

[0107] RgnTDC demonstrated the most robust activity across the library of select indoles tested (Fig. 5). Specifically, RgnTDC proved to have the highest production for nearly all substrates tested including: 2-chloro, 2-methyl, 6-cyano, 7-cyano, 7-iodo, 7-methoxy, and 7- nitro derivatives. Within this list, the 2-chloro, 2-methyl, 6-cyano, 7-cyano, and 7-nitro derivatives represent substrates not processed by PsiD in initial testing, while 7-iodo and 7- methoxy demonstrated enhanced yields over previously observed PsiD performance. RgnTDC represents the only decarboxylase tested to date which is capable of decarboxylation of 6- cyanotryptophan and 7-cyanotryptophan.

[0108] CroTDC demonstrated measurable activity towards all substrates tested with the exception of 6-cyanotryptophan and 7-cyanotryptophan (Fig. 6). Of the successful cases, only the 6-methoxy derivative outperformed RgnTDC on a direct comparison. The highest tryptamine titers were observed for the 2-methyl derivative and although, on an average, it falls a bit short of RgnTDC performance, further evaluation in extended pathways or under alternative fermentation conditions may result in enhanced performance.

[0109] PsmH demonstrated low, but measurable, activity for a number of substrates tested (Fig. 7), but no examples were able to outperform that of RgnTDC and CroTDC. It is important to note that PsmH does demonstrate some substrate selectivity not observed by RgnTDC, CroTDC, and PsiD. Specifically, PsmH does not use the natural amino acid tryptophan as a substrate, where all other decarboxylases tested do show this activity (Figure 7). This is specifically important in biological systems as tryptophan is generally present and tryptamineis frequently an unwanted side product which reduces overall product yield and can lead to reduced cell fitness by reducing tryptophan availability for protein synthesis.

[0110] Building off of this success, RgnTDC was incorporated with the full psilocybin biosynthesis pathway using a tripartite culture (tri-culture) system to enable the proof-of- principle biosynthesis of never before synthesized psilocybin derivatives. Using this approach, 3 novel psilocybin derivatives were produced including: 7-cyanopsilocybin (Figure 9), 7- ethylpsilocybin (Figure 10), and 7-carboxaldehydepsilocybin (Figure 11). To our knowledge this is the first report of the synthesis of these non-natural psilocybin derivatives.

[0111] Tri-culture experiments primarily used the previously reported Psilocybin Production Strain (pSilol6), the previously reported BL21 Star™(DE3) pETM6-SDM2X-PsiH-PcCPR strain (JF06 or PsiH strain), and the BL21 Star™(DE3) pETM6-SDM2X-T7-RgnTDC strain to enable psilocybin derivative production. Tri-culture experiments were also performed that used the pNor and the PsiH strain for norbaeocystin derivative production, along with a RgnTDC strain and the PsiH strain for 4-hydroxytryptamine derivative production. Overnight cultures were incubated at 37°C in AMM without MOPS, with ampicillin (80 mg / L), and were inoculated from freezer stocks. Experimental cultures were incubated in 48-well plates using 2 mL of media per well or in 125 mL Erlenmeyer flasks with 15 mL working volumes at 30°C. The production media was supplemented with 5 g / L of serine and 4.5 g / L methionine to enhance pathway performance. The 2% total inoculum was divided between the psilocybin production strain, the PsiH strain, and the RgnTDC strain at a ratio of 1 :8: 1 respectively, unless otherwise noted. Indole derivatives were added at inoculation to each well, except the negative (no indole) control, at 100 mg / L using 5 pL from a 40 mg / mL stock in EtOH. All haloindoles and other indoles which caused significant growth impairment were added 6 h after inoculation at 50 mg / L final concentration. Cultures were induced at 4 h using 1 mM IPTG. Samples were taken at 24 h for 48-well plate experiments, while flask experiments were sampled at 24, 48, and 72 h, and analyzed on LC-MS as described below.

[0112] 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 LC-MS analysis. Substitutedindole standards were created by addition of substituted indoles to AMM at concentrations of 100 mg / L.

[0113] Quantification of aromatic metabolites was performed using absorbance at 280 nm from the DAD and the metabolites were separated using an Agilent Zorbax Eclipse XDB-C18 analytical column (3.0 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. The mobile phase changes were implemented as linear gradients culminating at the stated values at the specified times. This method resulted in the following observed retention times as verified by analytical standards (when commercially available) and MS analysis (as described below): 4-hydroxyindole (6.6 min), 4-hydroxytryptophan (3.4 min), 4-hydroxytryptamine (3.2 min), norbaeocystin (1.6 min), baeocystin (1.9 min), and psilocybin (2.2 min).

[0114] Liquid chromatography mass spectrometry (LC-MS) data was collected where the full MS scan was used to provide an extracted ion chromatogram (EIC) for our compounds of interest. 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 550 °C, ion transfer tube temperature of 300 °C, source voltage of 3049 V, and source current of 15.90 pA.Example #3 : Evaluation of new Tryptophan Synthases expand and enhance biocatalytic capabilities

[0115] Here, we have evaluated the ability of three engineered standalone tryptophan synthases (Tri, Q90, and Azul) for the ability to alleviate biosynthetic bottlenecks when producing non-natural psilocybin and DMT derivatives. We started by evaluating the promiscuity of these enzymes in vivo, compared to the empty vector control strains only expressing basal levels of the native A. coli tryptophan synthase (TrpS or TrpB). We identified varied and expanded substrate promiscuity, opening up the biosynthetic possibilities beyond that of the wildtype E. coli variant.

[0116] Q90 demonstrated the broadest range of activities with significantly enhanced yields particularly at the 4, 5, and 6 position, enabling the production of multiple tryptophanderivatives not previously observed including: 4-cyano, 2-hydroxy, 5-iodo, 5-nitro, 6-nitro, 4- trifluoromethyl, and 5- trifluoromethyl, among others (Fig. 11). Tri demonstrated a broad range of activities with significantly enhanced yields particularly at the 4-position, however, did not demonstrate much improvement over the Q90 strain in initial studies (Fig. 12). Despite offering significant advantages over the E. coli wildtype variant, Azul showed limited advantages over Q90 and Tri, with the notable exception of the 1 -methyl derivative (Fig. 13). No further 1- position indole derivatives were considered in initial testing; however, efforts are underway to evaluate the hypothesis that Azul may serve as a 1 -position specialist, opening up a range of non-natural derivatives not practically achievable with other approaches.Example #4: Novel combination of tryptophan synthases and decarboxylases enable synthesis of new tryptamines

[0117] Combining the knowledge gained through Example 2 and Example 3 above, we attempted to biosynthesize tryptamines which had not previously been achievable due to limitations in either the A. coli tryptophan synthase, TrpB, or decarboxylase, PsiD. This proof of principle study demonstrated the production of 6-carboxaldehydetryptamine, 5- nitrotryptamine, 6-nitrotryptamine, 6-cyanotryptamine, 2-chloroctryptamine, 5- iodotryptamine, and 2-methyltryptamine through the use of tryptophan synthase Q90 and decarboxylase RgnTDC. To facilitate easy screening, this proof-of-principle study was performed using a co-culture strategy where strains expressing Q90 and RgnTDC, respectively, were grown together in a single fermentation vessel. This approach enables the cross sharing of metabolic products with the qualitative results presented in Fig. 14.Example #5: Evaluation of Halogenase Enzymes

[0118] Tryptophan modifying enzymes capable of halogenation, alkylation, and nitration are found in a variety of organisms (Neubauer et al., 2020; Sundberg, 1970). The high electron density indole ring is positioned on tryptophan in such a way what allows for regioselective electrophilic substitutions at the 5, 6, and 7 positions of tryptophan (Alkhalaf & Ryan, 2015). Halogenation has been an area of interest in pharmaceutical development due to its capabilities of creating drugs with desirable physiochemical properties for the treatment of numerous diseases (Sana et al., 2022). Some of these properties include increased passage of peptide drugs through the blood brain barrier (BBB) (Gentry et al., 1999), enhanced binding affinity of peptides to their target proteins (Rosa et al., 2015), and reduction of dangerous side-effects(Pescatore et al., 2015). That being said, halogenation can often increase cytotoxicity, especially for cancer cell applications that may benefit from that effect, however, this characteristic may not be suitable for the purpose of psychedelic medicine and should be considered if halogenated compounds were to be used in animal or human trials (Lindenblatt et al., 2019; Sana et al., 2022).

[0119] Chlorination enzymes are flavin-dependent halogenases that require FADH2 cofactor regeneration for efficient catalytic activity (Neubauer et al., 2020). An NAD(P)H flavin reductase partner is needed to reduce FADH2 to FAD to allow for sufficient co-factor availability, which in turn, increases enzymatic activity (Domergue et al., 2019; Dong et al., 2005). However, environments with high concentrations of FAD can lead to the accumulation of H2O2, as it is a product of the aerobic oxidation of FADH2, therefore altering the stability and activity of the enzymes within the system (Hou et al., 2023). That being said, a catalase can be used to prevent the buildup of this detrimental chemical within the cell (Ismail et al., 2019; Paul et al., 2014). The three-dimensional structure of tryptophan halogenases indicates the presence of two binding domains, a flavin binding domain and a substrate binding domain which are connected by a tunnel (Dong et al., 2005). The tunnel provides a connection between the isoalloxazine ring of FAD on one end and the tryptophan substrate on the other end therefore, the halogenating agent must have the capability of diffusing across this distance (Van Pee, 2012). Regiospecificity of tryptophan halogenases is made possible by interactions between tryptophan and the enzyme. Specifically, with 7-postion halogenases, the indole ring of tryptophan is tightly positioned between aromatic amino acids W455 and F103, the indole nitrogen forms a hydrogen bond with oxygen from a nearby peptide bond between E346 and S347, and the resulting structure interacts with amino acid side chains to demobilize tryptophan and place the desired halogenated position towards the tunnel (Van Pee, 2012). The minimal movement of the substrate ensures that only the specified position can be accessed by a chlorine ion (CF).

[0120] Tryptophan halogenases work to halogenate the free amino acid tryptophan through a reaction between O2 and FADH2 (Ortega et al., 2017). The oxidized product is a C4a- hydroperoxyflavin that reacts with a nearby bound chloride ion to produce hypochlorous acid (HOC1) (Shepherd et al., 2016; Van Pee, 2012; Yeh et al., 2007; Zhu et al., 2009). While HOC1 is necessary for halogenation, high concentrations can negatively impact the amino acid residues along the tunnel (Van Pee, 2012). That being said, this issue can be addressed by a serine residue (S347), which lies between the isoalloxazine ring and tryptophan, and its abilityto guide hypohalous acid, a byproduct of the enzyme reaction method, through the tunnel and redirect H0C1 to Lys79 and Glu346, a lysine and glutamate residue, respectively (Van Pee, 2012). The interaction between HOC1 and the two amino acids is necessary as this step places the chlorine ion from HOC1 towards the desired halogenated position of the substrate (Van Pee, 2012).

[0121] Results: Amino acid sequences for the halogenase genes, Streptomyces rugosporus PyrH (A4D0H5), Streptomyces toxytricini SttH (E9P162), Streptomyces violaceusniger ThHal (A0A1L1QK36), and Lentzea aerocolonigenes RebF (Q8K176) were sourced from Uniprot.

[0122] The halogenase genes and flavin reductase were codon-optimized for expression in E. coli. A PCR-based approach was used to amplify the genes with Ndel and Xhol flanking restriction sites. Studies have shown that a Q160N mutation to the PyrH gene can improve halogenation activity (Sana et al., 2022), therefore it was included in the synthetized gene. These products were ligated into a pETM6-SDM2x vector with a T7 promoter and transformed into DH5a competent cells. The resulting sequences were confirmed using whole-plasmid DNA sequencing.

[0123] Prior to incorporating each halogenase gene with the flavin reductase gene, some additional cloning was completed to further optimize the system. Due to the improved performance of the H10 promoter in some biosynthetic applications over consensus T7 (Adams et al., 2019; Flower et al., 2023a; Jones et al., 2015), all of the halogenase genes and flavin reductase gene were ligated into a pETM6-H10 vector using Ndel and Xhol. Using the ePathBrick standard assembly method, pETM6-H10-PyrH, pETM6-H10-SttH, and pETM6- HlO-ThHal were each combined in operon configuration with pETM6-H10-RebF vectors resulting in pETM6-H10-PyrH-RebF, pETM6-H10-SttH-RebF, and pETM6-H10-ThHal- RebF. Restriction digestion confirmed the successful incorporation of each vector into their respective constructs.

[0124] While there are many tryptophan halogenases with a variety of attributes, this thesis focuses on four chlorination enzymes and one flavin reductase. Previous work has shown success of site-specific tryptophan halogenase activity in bacteria, including: 5-position PyrH (Zehner et al., 2005), 6-position SttH (Zeng and Zhan, 2011), and ThHal (Moritzer et al., 2019), as well as flavin reductase gene, RebF (Sana et al., 2022). PmA (Dong et al., 2005) and RebH (Hou et al., 2023; Sana et al., 2022) are 7-position halogenases which demonstrated limited activity and unquantifiable amounts of chlorinated tryptophan in an assay for chlorination usingtryptophan substrate (Sana et al., 2022). However, recently published studies carried out in bacteria have shown successful activity of RebH (Hou et al., 2023).

[0125] Upon successful cloning of each halogenase and halogenase-reductase module each were transformed into BL21star™(DE3) for in vivo activity verification. Initial 48-well plate (2 mL working volume) assays were conducted to quantify 5-, 6-, and 7 -chlorotryptophan production from tryptophan. These small-scale studies require a source of tryptophan and chlorine which was provided by Andrew’s Magic Media (AMM), containing approximately 50 mM chlorine (Jones et al., 2015) with 1 g / L (5 mM) external tryptophan supplement. A singlesubstituted monoculture with the Q90 tryptophan synthase supplemented with 50 mg / L of 7- chloroindole was used as a positive control for the production of 7-chlorotryptophan. While 7- chlorotry ptophan has the same mass at 5-chlorotryptophan, the slight misalignment of retention times may be due to the different position of chlorine on the indole ring of tryptophan. PyrH and ThHal, with RebF, and RebH with and without RebF were able to produce quantifiable and detectable products by HPLC and LCMS while the chlorotryptophan derivatives produced by PyrH, ThHal without RebF, and SttH with and without RebF were below the limit of quantification by HPLC but detectable by LCMS. The 5-position halogenase construct, pETM6-H10-PyrH-RebF, outperformed all others, producing a chlorotryptophan peak area of 1.80 ± 0.15 mAU*min (Fig. 16). Constructs that produced quantifiable chlorotryptophan, showed statistically significant reduction in tryptophan concentration (p-value < 0.05).

[0126] The success of this proof-of-principle prompted the study of the activity of each halogenase module on 4-hydroxytryptophan, an intermediate in the psilocybin production pathway. This was accomplished by feeding 50 mg / L of 4-hydroxyindole to the fermentation media containing 1 g / L serine and 1 g / L methionine. A single-substituted monoculture with the TrpB tryptophan synthase supplemented with 50 mg / L of 7-chloro, 4-hydroxyindole was used as a positive control for the production of 7-chloro, 4-hydroxytryptophan. The misalignment of retention times is mostly likely due to the same conclusion previously discussed, namely, the same molecular weight but different position of the halogen on tryptophan. Chlorinated 4- hydroxytryptophan was achieved by the RebH halogenase alone, as well as the PyrH and RebH halogenase-reductase configurations. Although pETM6-H10-PyrH-RebF yielded the largest peak area of 0.60 ± 0.03 mAU*min, it represents an approximate 10-fold decrease in effective activity from the same constructs using a nonderivatized tryptophan substrate (Fig. 17A-C). This result suggests significant steric hindrances are encountered by the additional 4-hydroxygroup, which will limit the application of these wildtype enzymes in chloropsilocybin biosynthesis applications.

[0127] Further expanding upon this work, these halogenases were evaluated in a modified media which removed all sources of chloride and replaced with bromide to facilitate the bromination of tryptophan. As seen in Fig. 18, PryH and ThHal demonstrated the highest production of bromotryptophan at bromide concentrations of 125 and 150 mM, respectively. RebH and SttH bromonation activity was also observed, although overall production was low.

[0128] Example #6: Evaluation of Prenyltransferase Enzymes

[0129] Three prenyltransferase enzymes (5DMATS, PriB, and 7DMATS) were evaluated for their ability to act on the psilocybin pathway intermediate, 4-hydroxytryptophan. As seen in Figure 19, all three enzymes showed promiscuous activity towards this compound, producing the 4OH-dimethylallyltryptophan product with 289 m / z with an approximate retention time of 7.5 minutes. This opens the door for the production of various dimethylallyl-substituted psilocybin derivatives through leveraging the flexibility of the psilocybin pathway as documented in previous examples, above.

[0130] What has been described above includes examples of one or more embodiments. It is, of course, not possible to describe every conceivable modification and alteration of the above devices or methodologies for purposes of describing the aforementioned aspects, but one of ordinary skill in the art can recognize that many further modifications and permutations of various aspects are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims. Furthermore, to the extent that the term “includes” is used in either the details description or the claims, such term is intended to be inclusive in a manner similar to the term “comprising” as “comprising” is interpreted when employed as a transitional word in a claim.

[0131] Reference is made to one or more articles, patents, patent applications, or other publications, the entire content of which are expressly incorporated herein as if recited in their entirety.

Claims

CLAIMSWhat is claimed:

1. A method for the production of a non-naturally occurring tryptophan, a non-naturally occurring tryptamine, or a non-naturally occurring psilocybin, comprising administering a substrate to a cell culture, the cell culture comprising a recombinant prokaryotic cell having enzymatic activity selected from hydroxylase activity, decarboxylase activity, kinase activity, N-m ethyl transferase activity, and a prokaryotic cell having enzymatic activity selected from tryptophan synthase activity, halogenase activity, prenyltransferase activity, and combinations thereof.

2. The method of claim 1, wherein said hydroxylase activity is supplied by an enzyme selected from one or both of PsiH and CPR.

3. The method of claim 1, wherein said decarboxylase activity is supplied by an enzyme selected from RgnTDC, CroTDC, PsmH, PsiD, and combinations thereof.

4. The method of claim 1, wherein said kinase activity is supplied by PsiK.

5. The method of claim 1, wherein said tryptophan synthase activity is supplied by an enzyme selected from TrpB, Tri, Q90, Azul, and combinations thereof.

6. The method of claim 1, wherein said halogenase activity is supplied by an enzyme selected from PyrH, SttH, ThHal, and combinations thereof.

7. The method of claim 1, wherein said A-methyltransferase activity is supplied by PsiM.

8. The method of claim 1, wherein said enzymatic activity is at least two of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthase activity, halogenase activity, prenyltransferase activity, and / f-m ethyl transferase activity.

9. The method of claim 1, wherein said enzymatic activity is at least three of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthaseactivity, halogenase activity, prenyltransferase activity, and 7V-m ethyl transferase activity.

10. The method of claim 1, wherein said enzymatic activity is at least four of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthase activity, halogenase activity, prenyltransferase activity, and / f-m ethyl transferase activity.

11. The method of claim 1, wherein said enzymatic activity is at least five of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthase activity, halogenase activity, prenyltransferase activity, and / f-m ethyl transferase activity.

12. The method of claim 1, wherein said enzymatic activity is at least six of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthase activity, halogenase activity, prenyltransferase activity, and / f-m ethyl transferase activity.

13. The method of claim 1, wherein said enzymatic activity is each of hydroxylase activity, decarboxylase activity, kinase activity, tryptophan synthase activity, halogenase activity, prenyltransferase activity, and / f-m ethyl transferase activity.

14. The method of any preceding claim wherein said enzyme is endogenous to said cell.

15. The method of any preceding claim wherein said enzyme is supplied by an expression vector supplied to said cell.

16. The method of any preceding claim wherein said substrate is selected from an indole, a derivatized indole, a tryptophan, a derivatized tryptophan, a tryptamine, a derivatized tryptamine, or combinations thereof.

17. The method of claim 16 wherein said derivatized indole comprises a functional group substitution said functional group being selected from acyl halide, aldehyde, alkene, alkyne, amide, carboxamide, amino, azide, boronate, bromo, carboxylic acid, chloro, cyanate, cyano, ester, ethyl, fluoro, hydroxy, iodo, isocyanate, isopropyl, isothiocyanate, methoxy, methyl, nitrate, nitrite, nitro, propyl, prenyl, sulfinic acid,thiocyanate, thial, thiol, trifluoromethyl, trifluoromethoxy, butyl, and combinations thereof.

18. The method of claim 17 wherein the functional group substitution is at a position selected from position 1, position 2, position 4, position 5, position 6, position 7 of the indole ring, and combinations thereof.

19. The method of claim 16 wherein said derivatized tryptophan comprises a functional group substitution, said the functional group being selected from acyl halide, aldehyde, alkene, alkyne, amide, carboxamide, amino, azide, boronate, bromo, carboxylic acid, chloro, cyanate, cyano, ester, ethyl, fluoro, hydroxy, iodo, isocyanate, isopropyl, isothiocyanate, methoxy, methyl, nitrate, nitrite, nitro, propyl, prenyl, sulfinic acid, thiocyanate, thial, thiol, trifluoromethyl, trifluoromethoxy, butyl, and combinations thereof.

20. The method of claim 19 wherein said chemical substitution is at a position selected from position 1, position 2, position 4, position 5, position 6, position 7, and combinations thereof.

21. The method of claim 16 wherein said derivatized tryptamine comprises a functional group substitution, said functional group being selected from acyl halide, aldehyde, alkene, alkyne, amide, carboxamide, amino, azide, boronate, bromo, carboxylic acid, chloro, cyanate, cyano, ester, ethyl, fluoro, hydroxy, iodo, isocyanate, isopropyl, isothiocyanate, methoxy, methyl, nitrate, nitrite, nitro, propyl, prenyl, sulfinic acid, thiocyanate, thial, thiol, trifluoromethyl, trifluoromethoxy, butyl, and combinations thereof.

22. The method of claim 21 wherein said chemical substitution is at a position selected from position 1, position 2, position 4, position 5, position 6, position 7, and combinations thereof.

23. The method of any preceding claim wherein said cell culture is a co-culture comprising at least two cell strains, or at least three cell strains, at least four cell strains, or at least five cell strains.

24. An expression vector comprising at least one gene selected from RgnTDC, CroTDC, 5DMATS, PriB, 7DMATS, Tri, Q90, Azul, PyrH, SttH, ThHal, RebF, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration, optionally wherein each promoter is independently selected from the group consisting of G6 mutant T7, H9 mutant T7, Hl 0 mutant T7, C4 mutant T7, consensus T7, Lac, LacUV5, tac, trc, GAP, and xylA promoter.

25. A culture comprising a cell strain selected from a. a first recombinant prokaryotic cell comprising an enzyme having hydroxylase activity, said enzyme being selected from one or both of PsiH and CPR; b. a second recombinant prokaryotic cell comprising an enzyme having decarboxylase activity, said enzyme being selected from RgnTDC, CroTDC, PsmH, PsiD, and combinations thereof; c. a third recombinant prokaryotic cell comprising an enzyme having kinase activity, said kinase activity being supplied by PsiK; d. a fourth recombinant prokaryotic cell comprising an enzyme having tryptophan synthase activity, said enzyme being selected from Tri, Q90 and Azul, and combinations thereof; e. a fifth recombinant prokaryotic cell comprising an enzyme having halogenase activity, said enzyme being selected from PyrH, SttH, ThHal, and combinations thereof; f. a sixth recombinant prokaryotic cell comprising an enzyme having N- methyltransferase activity, said A-m ethyl transferase activity being supplied by PsiM; and g. a seventh recombinant prokaryotic cell comprising an enzyme having prenyltransferase activity, the prenyltransferase activity being supplied by 5DMATS, PriB, 7DMATS, and combinations thereof.

26. A co-culture comprising at least two cell strains, said at least two cell strains, collectively having enzymatic activity of at least two, at least three, at least four, at least five, at least six, or all seven of the following: a. hydroxylase activity; b. decarboxylase activity; c. kinase activity; d. tryptophan synthase activity; e. halogenase activity; f. A-m ethyl transferase activity; and g. prenyltransferase activity.

27. A recombinant prokaryotic cell comprising one or more expression vector comprising a gene selected from of RgnTDC, CroTDC, PsmH, Tri, Q90, Azul, PyrH, SttH, ThHal, RebF, 5DMATS, PriB, 7DMATS, and combinations thereof in combination with at least one of PsiH, CPR, PsiD, PsiK, PsiM and combinations thereof.

28. The recombinant prokaryotic cell of claim 27, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli. Corynebacterium glutamicum, Vibrio natriegens, Bacillus sublilis. Bacillus megalerium. Escherichia coli Nissle 1917, Clostridium acelobullyicum. Streptomyces coelicolor. Lactococcus laclis. Pseudomonas putida, Streptomyces clavuligerus. and Streptomyces venezuelae.

29. A composition comprising 7-cyanopsilocybin, having the structure:

30. A composition comprising 7-ethylpsilocybin, having the structure:

31. A composition comprising 7-carboxaldehydepsilocybin, having the structure:

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