WO2022226493A1

WO2022226493A1 - Optimized methods for the production of psilocybin and intermediates or side products

Info

Publication number: WO2022226493A1
Application number: PCT/US2022/071797
Authority: WO
Inventors: John Andrew Jones; Philip John O'DELL
Original assignee: Miami University
Priority date: 2021-04-19
Filing date: 2022-04-19
Publication date: 2022-10-27

Abstract

Provided are methods, host cells, transposons, transposomes, and kits for the optimized production of psilocybin or an intermediate or a side product thereof in a prokaryotic host cell. Also provided are methods, host cells, transposons, transposomes, and kits for the optimized production of norbaeocystin in a prokaryotic host cell. 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

OPTIMIZED METHODS 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 optimized methods 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. 63/176,590, filed April 19, 2021, and U.S. Provisional Application No.

63/263,611, filed November 5, 2021, each of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which is submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 7, 2022, is named 315691-00020_Sequence_Listing.txt and is 22,377 bytes in size.

BACKGROUND

[0004] Nearly one in five Americans live with some form of mental illness. Psilocybin and other psychedelics have shown promising initial clinical trials for therapeutic use in the treatment of depression, anxiety, addiction, and post traumatic stress disorder.

[0005] Because of its potential for treatment for a number of anxiety and mental-health related conditions, interest in psilocybin is significant. However, due to roadblocks in methods of obtaining drug product (synthesis and/or extraction from a known biological source), supply is not currently able to meet the growing demand.

[0006] Psilocybin (4-phosphoryloxy-/V,/V-di methyl tryptaminc) has gained attention in pharmaceutical markets as a result of recent clinical studies. (Robin Carhart-Harris, et ah, 2021) 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.

[0007] Psilocybin was first purified from the Psilocybe mexicana mushroom by the Swiss chemist, Albert Hoffmann, in 1958. The first reports of the complete chemical synthesis of psilocybin were published in 1959; however, large-scale synthesis methods were not developed until the early 2000’ s by Shirota and colleagues at the National Institute of Sciences in Tokyo. Despite significant improvements over early synthetic routes, current methods remain tedious and costly, involving numerous intermediate separation and purification steps resulting in an overall yield of 49% from 4-hydroxyindole, incurring an estimated cost of $2 USD per milligram for pharmaceutical-grade psilocybin.

[0008] Much of the interest in psilocybin is due to its biosynthetic precursors — norbaeocystin and baeocystin. These compounds have structural similarity to the neurotransmitter serotonin and sparked the interest of researchers who were curious to understand the mechanism behind their hallucinogenic properties. After being named a Schedule I compound in the US with implementation of the Controlled Substance Act of 1970, research efforts involving psilocybin were abandoned for other less regulated bioactive molecules; however, experts in the field have suggested a reclassification to schedule IV would be appropriate if a psilocybin-containing medicine were to be approved in the future.

[0009] Clinical trials with psilocybin as a medication for individuals struggling with treatment- resistant depression are ongoing.

[0010] Recently, psilocybin has been produced on a gram per liter scale in E. coli using 4-OH indole as a substrate. (Adams et al., 2019).

[0011] Most bacterial chemical production platforms utilize plasmid vectors for gene expression due to the ease of cloning and optimization. However, these vector-based systems necessitate costly antibiotics for genetic stability, making many processes less financially viable and potentially increasing the prevalence of antibiotic resistance in the environment. Additionally, the expression of the antibiotic resistance cassette as well as plasmid replication and maintenance siphons metabolic flux away from cell growth or heterologous enzyme expression, therefore lowering production of the product of interest (Wu, G. et ah, 2016). Cells can also mutate plasmid DNA, which can alleviate metabolic burden of the pathway expression while still maintaining antibiotic resistance, leading to significant portions of biomass becoming unproductive in a fermentation (Wu, G. et ah, 2016).

[0012] There remains a need for optimized methods for the production of psilocybin and intermediates or side products thereof.

SUMMARY

[0013] The general inventive concepts relate to and contemplate methods and compositions for producing psilocybin or an intermediate or a side product thereof.

[0014] 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 transposomes, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell. 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.

[0015] In some embodiments, the transposome comprises (a) a transposon comprising a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof, and (b) a transposase.

[0016] In some embodiments, the transposase is Tn5, Ac, Spm/En, Tel, Tc3, Pogo, Slide, Tpnl, Tgml, Taml, Tam3, a P element, Pad, Hobo or PiggyBac. In further embodiments, the transposase is a member of the Tnrl/Stowaway family, a member of the En/Spm family, a member of the Tourist family, a member of the broad Tc 1/mariner superfamily, a member of the Minos family, a member of the Tn5 family, a Tn relative, a member of the Mutator family, a member of the miniature inverted-repeat transposable element (MITE) class, a member of the Basho family, a member of the P-element family, a member of the Sleeping Beauty family, a member of the Pogo family, a member of the Tiggers family, a member of the broad class of bacterial IS elements, a member of the broad hAT-transposable element superfamily, a member of the Hermes family, a member of the Mosl family, or a member of the PiggyBac family. In some embodiments, the transposase is engineered.

[0017] In some embodiments, the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4- hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT). In some embodiments the intermediate of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, or 4-hydroxytryptamine. In some embodiments, the side product of psilocybin is aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).

[0018] In some embodiments, the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine and combinations thereof. In further embodiments, the supplement is fed continuously to the host cell.

[0019] In some embodiments, the host cell is grown in an actively growing culture.

[0020] In some embodiments, the method further comprises use of transcriptional balancing on the prokaryotic host cell genome to enhance specifically psilocybin, an intermediate, or a side product thereof. In some embodiments, the transcriptional balancing is achieved using a promoter 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. In some embodiments, the transcriptional balancing is mediated through site specific Cas9 counterselection against an unoptimized construct.

[0021] Also provided is a recombinant prokaryotic cell comprising one or more transposomes, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

[0022] Also provided is a recombinant prokaryotic cell comprising one or more transposons, wherein each transposon comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof. [0023] Also provided is a recombinant prokaryotic cell comprising a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof, wherein the psilocybin production gene(s) is integrated into the genome of the prokaryotic cell. In some embodiments, a psiD gene, a psiK gene, and a psiM gene are all under control of a single promoter in operon configuration. 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. In some embodiments, a psiD gene, a psiK gene, and a psiM gene are each under control of a separate promoter in pseudooperon or monocistronic configuration. 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.

[0024] Provided is a transposome for introducing at least one gene associated with psilocybin production; the gene may be selected from: psiD, psiK, and psiM and combinations thereof.

[0025] Provided is a transposome comprising a psiD gene, a psiK gene, and a psiM gene all under control of a single promoter in operon configuration. 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.

[0026] Provided is a transposome comprising a psiD gene, a psiK gene, and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. 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.

[0027] Provided is a transposon for introducing at least one gene associated with psilocybin production; the gene may be selected from: psiD, psiK, and psiM and combinations thereof.

[0028] Provided is a transposon comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. 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. [0029] Provided is a transposon comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. 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.

[0030] Also provided is a transfection kit comprising a transposon according to any of the embodiments described herein. In some embodiments, the transfection kit further comprises a transposase.

[0031] In some aspects of any one of the embodiments described herein, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 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.

[0032] In some aspects of any one of the embodiments described herein, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 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.

[0033] In some aspects of any one of the embodiments described herein, the psiM gene encodes a polypeptide comprising the amino acid sequence of 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.

[0034] Provided is a method for the production of norbaeocystin comprising contacting a prokaryotic host cell with one or more transposomes, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof; and culturing the host cell. In certain embodiments, none of the transposomes comprises psiM. 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.

[0035] Also provided is a recombinant prokaryotic cell comprising one or more transposomes, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof. In some embodiments, the transposome comprises a psiD gene and a psiK gene gene all under control of a single promoter in operon configuration. 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. In some embodiments, the transposome comprises a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. 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.

[0036] Also provided is a recombinant prokaryotic cell comprising one or more transposons, wherein each transposon comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof. In some embodiments, the transposon comprises a psiD gene and a psiK gene gene all under control of a single promoter in operon configuration. 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. In some embodiments, the transposon comprises a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. 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.

[0037] Also provided is a recombinant prokaryotic cell comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, wherein the psilocybin production gene(s) is integrated into the genome of the prokaryotic cell. In some embodiments, a psiD gene, and a psiK gene are all under control of a single promoter in operon configuration. 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. In some embodiments, a psiD gene, and a psiK gene are each under control of a separate promoter in pseudooperon or monocistronic configuration. 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.

[0038] Provided is a transposome for introducing at least one gene associated with psilocybin production; the gene may be selected from: psiD, psiK, and combinations thereof. In some embodiments, the transposome comprises a psiD gene and a psiK gene gene all under control of a single promoter in operon configuration. 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. In some embodiments, the transposome comprises a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. 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] Provided is a transposon for introducing at least one gene associated with psilocybin production; the gene may be selected from: psiD, psiK, and combinations thereof. In some embodiments, the transposon comprises a psiD gene and a psiK gene gene all under control of a single promoter in operon configuration. 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. In some embodiments, the transposon comprises a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. 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.

[0040] Also provided is a transfection kit comprising a transposon according to any of the embodiments described herein. In some embodiments, the transfection kit further comprises a transposase. [0041] In some aspects of any one of the embodiments described herein, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 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.

[0042] In some aspects of any one of the embodiments described herein, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 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.

DESCRIPTION OF THE FIGURES

[0043] FIG. 1 shows psilocybin biosynthesis pathway consisting of three heterologous enzymes, PsiD, PsiK, and PsiM, and highlighting the media supplements of serine and methionine. PsiD: L-tryptophan decarboxylase; PsiK: kinase; PsiM: S-adenosyl-L-methionine (SAM)-dependent N-methyltransferase; TrpB: tryptophan synthase beta subunit; Ser: serine; Met: methionine; 1:4- hydroxy indole; 2: 4-hydroxytryptophan; 3: 4-hydroxytryptamine; 4: norbaeocystin;

5: baeocystin; 6: psilocybin.

[0044] FIG. 2 shows the T7 consensus promoter (SEQ ID NO: 11) alongside four T7 mutant promoters (C4 (SEQ ID NO: 10), H10 (SEQ ID NO: 7), H9 (SEQ ID NO: 9) and G6 (SEQ ID NO: 8)). Base-pair differences are shown in gray. All four mutant promoters have 2-3 differences in the PAM-proximal seed nucleation region (PPSNR) from the T7, making them good candidates for Cas9 assisted integration.

[0045] FIG. 3 illustrates that the five mutants that showed pathway activity had varying levels of metabolite concentrations. All mutants showed baeocystin buildup, showing a lack of activity by PsiM, the methyltransferase that converts baeocystin to psilocybin. This buildup was also observed in the positive control, indicating additional methionine supplement may be required in the fermentation media. The positive control was an E. coli strain expressing the psilocybin biosynthesis pathway, containing psiD, psiK, and psiM, from an episomal vector, controlled by the H10 mutant T7 promoter in operon configuration. [0046] FIG. 4 shows that the positive control produced approximately 100 mg/L psilocybin. The positive control was an E. coli strain expressing the psilocybin biosynthesis pathway, containing psiD, psiK, and psiM, from an episomal vector, controlled by the H10 mutant T7 promoter in operon configuration. The best performing mutant produced ~4 mg/L psilocybin, which is the first instance of psilocybin production from chromosomally integrated genes. Additionally, four other mutants showed pathway activity, as seen by the production of intermediate products, 4- hydroxytryptamine and baeocystin.

[0047] FIG. 5 shows that a wide range of growth rates were observed across the mutant library. Without wishing to be bound by theory, this may be due to the different integration locations of the psilocybin pathway. Mutants where the integration occurred in the middle of semi-essential genes showed much lower growth rates compared to the control. However, some mutants showed similar, though still lower, growth compared to the control. These mutants likely have the integration in the intergenic space in the chromosome.

[0048] FIG. 6 is a table showing the differences between chromosomal expression and vector- based expression.

[0049] FIGS. 7A-7C show a schematic of some of the methods described herein. FIG. 7A shows PCR amplification of Kan resistance cassette, restriction ligation cloning into pETM6-SDM2x, restriction ligation cloning of T7-PsiDKM into KpET, and PCR amplification of KanR and T7- PsiDKM with mosaic ends (ME) on primer tails. FIG. 7B is a continuation of FIG. 7 A and shows the fragment formed by PCR amplification, then shows gel extraction or Dpnl digestion and reaction with transposase (circles) to yield a transposome complex, which is then electroporated. FIG. 7C is a continuation of FIG. 7B and shows random integration of the transposome complex into the genome, and also shows Cas9 assisted promoter library creation.

[0050] FIG. 8 shows the “cut and paste” mechanism of the transposon.

[0051] FIG. 9 shows the Cas9 protein-sgRNA complex. The Cas9 protein attaches to the genomic DNA and, when the single guide RNA binds to its complement, a double strand break (DSB) is created, inducing cell death in E. coli if not rescued by a homologous recombination- based repair. [0052] FIG. 10 shows two plasmids used in the promoter swapping protocol. Left: pCas9-CR4 contains the Cas9 protein under control of the tetR bidirectional promoter and is chloramphenicol (Cm) resistant. Right: pKDsgRNA-T7 contains the single guide RNA (sgRNA) and sgRNA scaffolding targeted to the T7 consensus promoter necessary for site-specific counterselection also inducible by aTc. pKDsgRNA-T7 also contains the l-red recombinase system inducible by L-arabinose. The pKD vector has a temperature sensitive origin of replication and cultures containing the plasmid must be grown at 30 °C. Both maps are exported from SnapGene^®.

[0053] FIG. 11 shows a graphical representation of the genomic footprinting procedure.

[0054] FIGs. 12A-12B show psilocybin and norbaeocystin production, respectively. FIG. 12A shows the psilocybin production of integrated mutants: 2.7, 7.11, and 8.11 and the controls: the optimized vector, pETM6-SDM2X-H10-PsiDKM (pPsilol6 labeled “H10 vector”), and pETM6- SDM2X-T7-PsiDKM (labeled “T7 vector”). All cultures were tested in singlicate to increase the throughput of the HPLC-based screening method. Mutants that showed no psilocybin pathway activity are not shown. FIG. 12B shows the norbaeocystin production of the integrated mutant: 1.2, and the controls: in the optimized vector, pETM6-SDM2X-C4-PsiDK (pNor labeled “C4 vector”) as well as pETM6-SDM2X-T7-PsiDK (labeled “T7 vector”). All cultures were tested in singlicate to increase the throughput of the HPLC-based screening method. Mutants that showed no norbaeocystin pathway activity are not shown.

[0055] FIG. 13 shows norbaeocystin titers of selected integration mutants. The standard deviation of duplicate fermentations are represented by error bars. pETM6-SDM2X-C4-PsiDK (pNor labeled “C4 vector”) is the optimized expression vector, and pETM6-SDM2X-T7-PsiDK (labeled “T7 vector”) is the unoptimized expression vector containing the same T7 promoter as the integration mutants. Three of the mutants were selected for the in vivo transcriptional optimization protocol. Those mutants are Mutant 1.9, Mutant 3.18 and Mutant 1.6. Mutant 1.2 (not shown here) was generated previously and was also selected for the in vivo transcriptional optimization. [0056] FIG. 14 shows titers of the promoter swapped mutants. The T7 controlled mutants are shown as 1.2-T7 and 1.9-T7. Promoter mutants are shown as 1.2,xx or 1.9,xx depending on the parent mutant. The optimized norbaeocystin production strain is shown as C4 vector (pETM6- SDM2X-C4-PsiDK or pNor). The original mutants harboring the two vectors used in the transcriptional optimization protocol, pCas9CR4 and pKDsgRNA-T7, were also tested to assess if the presence of these plasmids affected production. These strains are shown as 1.2- T7+plasmids and 1.9-T7+plasmids.

[0057] FIG. 15 shows the E. coli genome and two locations of the pathway integrations. The locations are labelled with the name of the mutants (2.7, 8.11). The locations are also labelled with the names of the nearest genes as both integrations were found to be in intergenic space.

DETAILED DESCRIPTION

[0058] 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.

[0059] 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.

[0060] 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.

[0061] “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. [0062] 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.

[0063] 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.

[0064] As used herein, the term “prokaryotic host cell” means a prokaryotic cell that is susceptible to transformation, transfection, transduction, or the like, with a transposon, a transposome, 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.

[0065] 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.

[0066] As used herein, the term “intermediate” of psilocybin means an intermediate in the production or biosynthesis of psilocybin, e.g., norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine.

[0067] As used herein, the term “side product” of psilocybin means a side product in the production or biosynthesis of psilocybin, e.g., aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT). [0068] 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, and methods for the production of norbaeocystin.

[0069] Despite advances in the chemical synthesis of psilocybin, current methodologies struggle to provide sufficient material in a cost-effective manner. New advancements fueled Applicant’s interest in developing a more cost-effective and easily manipulated host for the biosynthetic production of psilocybin.

[0070] Chromosomally integrated pathways require no antibiotic selection for gene stability and ensure relative genetic homogeneity both within and between cultures, which can lead to lower costs and more consistent titers between cultures. However, chromosomal integration in bacteria has its challenges. The transcriptional landscape of the E. coli genome varies in transcriptional propensity, the amount of RNA produced per unit DNA, as much as four orders of magnitude depending on location (Scholz, S.A., et al. 2019). This represents a near infinite solution space, making traditional screening of the full potential library practically impossible for systems not designed with a high-throughput selection-based system. Additionally, it is not necessarily the case that the areas of highest transcriptional propensity will produce the highest titers, as evidenced by the optimal promoter in the vector-based psilocybin production strain of E. coli, H10 (Adams et al., 2019), a mutant T7 -lac promoter that has been characterized as having 40% - 70% of the strength of the consensus T7 promoter (Jones, J.A. et al. 2015). Beyond optimizing the location on the chromosome, simply integrating a fragment as large as the psilocybin pathway (~10kb: PsiD, PsiK, PsiM and a kanamycin resistance cassette) is difficult. The size of the construct is at or above the maximum integration fragment size for conventional methods that utilize a one-pot integration strategy (Posfai et al., 1999). Additionally, for methods that can theoretically integrate large fragments, integration efficiency decreases with increasing fragment size (Reisch & Prather, 2015). Although site-specific integration is useful when productive chromosomal sites have been identified, it’s use greatly reduces the solution space. Random integrations allow for screening of many chromosomal locations that vary in transcriptional propensity. Another issue with chromosomally integrated pathways is the difficulty in post integration genetic optimization such as promoter engineering, which requires site specific, scarless, and efficient genomic modifications to probe the solution space. [0071] The difficulties in chromosomal integration and optimization are to be overcome in this project with the goal of creating a generalizable approach for large metabolic pathway integration and subsequent transcriptional optimization. Genetically optimized strains containing the transcriptionally optimized, chromosomally integrated psilocybin and norbaeocystin pathways will be produced as proofs of principle.

Current Methods in Chromosomal Integration

[0072] Manipulating bacterial chromosomal DNA, especially E. coli, has been of interest in the recent decades for the reasons outlined above. There have been many techniques developed in this pursuit. Some methods utilize the phage-derived elements: integrase and excisionase. One such method, Conditional-Replication, Integration, Excision, and Retrieval (CRIM) plasmids allow for not only efficient integration of large constructs, but also excision of integrated fragments (Haldimann & Wanner, 2001). Another uses the One-Step Integration Plasmid, pOSIP, which allows for integrations up to 4.6 kb and efficient excision (St-Pierre et al., 2013). Both methods obtain efficient integration in a one-pot strategy but are limited to one of five attB sites in the E. coli chromosome.

[0073] The l-Red recombinase system is a genetic tool that consists of a three gene construct: gam , beta , and exo. It is used to achieve homologous recombination of small fragments into the chromosome. Gam prevents native E. coli endonucleases from degrading the linear integration fragment. Beta binds to the single stranded overhangs and the homologous chromosomal region and facilitates recombination. Exo degrades the double stranded fragment from the 5’ ends, leaving a 3’ overhang in the processed regions. Homology arms containing a sequence identical to the region surrounding the integration site are necessary for integration with homology length being proportional to integration efficiency. The l-Red recombinase system has been used extensively to make small scarless integrations (Reisch & Prather, 2015), to integrate larger fragments with a scar, and for gene deletions (Posfai et al., 1999) in one -pot reactions. It has also been used to integrate “landing pads” for use with other integration methods that are able to integrate larger fragments (Bassalo et al., 2016). In integration strategies utilizing phage-derived elements, where there are few choices for integration location, a small attB site can be integrated first, allowing for large pathway integration anywhere on the chromosome in a two-step integration approach. “Landing pads” can also house unique endonuclease cut sites. In one technique, 18 bp cut sites for I-Scel are inserted into the desired locus first. When I-Scel is expressed in vivo alongside Gam, Beta, and Exo, a vector also containing I-Scel cut sites is linearized and a DSB is achieved, which increases homologous recombination efficiency (Kuhlman & Cox, 2010), allowing for integrations of up to 9 kb.

[0074] Homologous recombination is often used in conjunction with flippase, which can remove sections of DNA in vivo that are flanked by flippase recognition target (FRT) sequences. A small construct can be site- specifically integrated into the chromosome containing a selection marker with the intent to insert a construct or inactivate an endogenous gene (Datsenko & Wanner, 2000). After counter- selection, flippase can be expressed in vivo to remove the selection marker for iterative rounds of integration.

[0075] The CRISPR/Cas9 system has also been used as a counter selective pressure to enrich mutant populations after homologous recombination (Bassalo et ah, 2016; Reisch & Prather, 2015). The Streptococcus pyogenes native protein Cas9 is able to achieve a double strand break (DSB) to a targeted sequence in the chromosome. A linear DNA fragment is provided to replace the targeted region, rescuing the strain from the Cas9-induced double strand break counterselection. The counter- selection can be targeted to nearly any location in the chromosome, and inserted fragments may be up to 10 kb (Bassalo et ah, 2016) using the l-red recombinase system without an antibiotic resistance cassette.

[0076] Transposon mediated integration has been used to make either random or site-specific chromosomal integrations in E. coli as well. Transposons can also be used in conjunction with the l-Red recombinase system. In the case of mini-Tn7 transposons, which require an attTn7 attachment site to make integrations, a strategy similar to phage-derived methods was used to integrate a “landing pad” with the appropriate attachment site prior to integration of the pathway (Roos, K et ah, 2015). Mini-mu transposons do not require a specific site for integration. They have been used to make random integrations (Lorenzo et ah, 1990) in the E. coli chromosome. The fragments have consisted of an antibiotic selection marker and a conditional origin of replication with a maximum size of 5.4 kb. [0077] Tn5 transposons allow for the most freedom in the integration of large fragments, making pseudorandom integrations in the chromosome with no reported limitations in the construct size. Tn5 transposons have been used to integrate an isobutanol pathway ((Saleski et al., 2021) in E. coli, improve sequencing processes (Kia et al., 2017), and obtain transcriptional propensity data on the E. coli chromosome (Scholz, S.A. et al., 2019).

Transposon Mechanism

[0078] Transposons act through a “cut and paste” mechanism, cleaving transposable elements (TEs) and inserting them randomly into double stranded DNA, including chromosomal, episomal, and plasmid DNA, in vivo and in vitro. Transposase is found endogenously in nearly all organisms, and accounts for the phenomenon of “gene jumping.” Tn5 transposase is a prokaryotic enzyme and has been subject to several mutations (Kia et al., 2017) for increased activity so that it can be used effectively in synthetic biology. Transposition in E. coli is controlled by two transposon protein encoding genes, Tnp, the active transposase gene and its truncated version, Inp, which acts as an inhibitor (Wiegand & Reznikoff, 1992). The transposase mechanism is shown in schematic form in FIG. 8. Tn5 transposase recognizes a specific sequence of inverted repeats, called mosaic ends (MEs), that flank the TE. It is to these MEs that two transposase subunits bind and cleave a TE from its backbone. The subunits then bind to one another, forming a synaptic complex, or transposome. When the transposome is near other double stranded DNA, TE is inserted into it leaving 9 bp scars flanking the integration. The transposome complex is stable at -20 °C for several months. After the transposase is reacted with the transposable DNA, it is can be electroporated into E. coli and produces high efficiency integrations in a pseudorandom manner, showing a slight bias for G/C rich regions (Green et al., 2012).

EXAMPLES

[0079] The following examples describe various compositions and methods for genetic modification of cells to aid in the production of psilocybin, according to the general inventive concepts. Example 1

Materials and Methods

[0080] A kanamycin resistance cassette was added to the pETM6-SDM2x vector by PCR and restriction cloning to create pETM6-SDM2x-FRT-Kan-FRT, or KpET. The psilocybin pathway, PsiDKM, in basic operonic form under the control of the T7 consensus promoter was restriction cloned into KpET to yield KpET-T7-PsiDKM. KpET-T7-PsiDKM was used as template to amplify the integration fragment, which is composed of the kanamycin resistance cassette and T7-PsiDKM. The PCR fragment was gel extracted and reacted with Tn5-EZ transposase from Lucigen (Middleton, Wisconsin, USA) to create a transposome. The transposome was electroporated into BL21star™(DE3), which, after outgrowth in SOC, was plated on LB agar plates with kanamycin. Colonies were screened for the template plasmid by verifying a lack of ampicillin resistance.

[0081] Colonies that were negative for ampicillin resistance were then inoculated in 2 mL of AMM with 350 mg/L 4-OH indole and no antibiotic from a preculture. Cultures were induced with 1 mM IPTG after 4 hours. Metabolite concentration was determined by HPLC analysis and optical density measurements were taken at 600 nm 48 hours after inoculation.

Results

[0082] A schematic of some of the methods described herein is illustrated in FIGS. 7A-7C. FIG. 7A shows PCR amplification of Kan resistance cassette, restriction ligation cloning into pETM6- SDM2x, restriction ligation cloning of T7-PsiDKM into KpET, and PCR amplification of KanR and T7-PsiDKM with mosaic ends (ME) on primer tails. FIG. 7B is a continuation of FIG. 7A and shows the fragment formed by PCR amplification, then shows gel extraction or Dpnl digestion and reaction with transposase (circles) to yield a transposome complex, which is then electroporated. FIG. 7C is a continuation of FIG. 7B and shows random integration of the transposome complex into the genome, and also shows Cas9 assisted promoter library creation.

[0083] The T7 consensus promoter is shown alongside four T7 mutant promoters in FIG. 2. Base-pair differences are shown in gray. All four mutant promoters have 2-3 differences in the PAM-proximal seed nucleation region (PPSNR) from the T7, making them good candidates for Cas9 assisted integration. [0084] The five mutants that showed pathway activity had varying levels of metabolite concentrations, as shown in FIG. 3. All mutants showed baeocystin buildup, showing a lack of activity by PsiM, the methyltransferase that converts baeocystin to psilocybin. This buildup was also observed in the positive control, indicating additional methionine supplement may be required in the fermentation media.

[0085] The positive control produced approximately 100 mg/L psilocybin, as shown in in FIG. 4. The best performing mutant produced approximately 4 mg/L psilocybin, which is the first instance of psilocybin production from chromosomally integrated genes. Additionally, four other mutants showed pathway activity, as seen by the production of intermediate products, 4- hydroxytryptamine and baeocystin.

[0086] A wide range of growth rates were observed across the mutant library, as illustrated in FIG. 5. Without wishing to be bound by theory, this may be due to the different integration locations of the psilocybin pathway. Mutants where the integration occurred in the middle of semi-essential genes showed much lower growth rates compared to the control. However, some mutants showed similar, though still lower, growth compared to the control. These mutants likely have the integration in the intergenic space in the chromosome.

[0087] The differences between chromosomal expression and vector-based expression are illustrated in the table in FIG. 6.

Example 2

Materials and Methods Strains and Media

[0088] E. coli DH5a was used for all DNA manipulation including plasmid propagation and transformations of ligated digest products. E. coli BL21 star™ (DE3) was used for all expression systems and was the target of all genomic integrations.

[0089] Luria Broth (LB) was used for all plasmid propagation and preparation of electrocompetent cells. Andrews Magic Media (AMM) without MOPS or tricine was used for all production experiments including the preculture. Building ofLaunchpad Vectors: pKET-PsiDK and pKET-PsiDKM

[0090] A kanamycin resistance cassette was PCR amplified from pKD4 with primers PI and P2. Both primers include restriction enzyme cut sites ( Nhel and Sail) for easy restriction ligation cloning into the pETM6-SDM2x vector to create pETM6-SDM2x-FRT-Kan-FRT, or pKET.

[0091] The psilocybin pathway PsiDKM consisting of PsiD, PsiK, and PsiM in basic operonic form under the control of the T7 consensus promoter (T7-PsiDKM) was restriction cloned from pETM6-SDM2x-T7-PsiDKM into pKET to yield pKET-T7-PsiDKM using Bad and Apa\. The same procedure was followed for the norbaeocystin pathway, beginning with pETM6-SDM2x- T7-PsiDK to yield pKET-T7-PsiDK.

Integration of Pathways

[0092] The launchpad vector pKET-T7-PsiDKM was used as template to amplify the integration fragment, which is composed of the kanamycin resistance cassette and T7-PsiDKM flanked on both sides by ME’s (CTGTCTCTTATACACATCT (SEQ ID NO: 42)) from the primer tails, with primers P3 and P4, which feature phosphate groups on the 5’ ends. The PCR fragment was gel extracted and reacted with Tn5-EZ transposase (Lucigen) according to the manufacturer’s protocol to create a 4 pL transposome solution. The first iteration of the method utilized a gel extraction protocol, resulting in a small library of productive strains due to the mutative effects of ethidium bromide and UV light. The second iteration of the method utilizes purification of the PCR without exposure to UV light or ethidium bromide. The transposome solution (1 pL) was electroporated into BL21 star™ (DE3), which, after outgrowth in SOC, was spread on LB agar plates with kanamycin. Colonies were screened for integration and a lack of template plasmid by streaking on a plate with ampicillin, a plate with kanamycin, and a plate with no antibiotics. Mutants positive for the integration and negative for the template plasmid grew on the kanamycin-containing plate and the plate without antibiotics and not on the plate with ampicillin.

[0093] pKET-T7-PsiDK was used as a template for PCR amplification of the integration fragment. The norbaeocystin pathway fragment was also reacted with Tn5-EZ transposase, electroporated, and screened in the same manner described above. Library Screening

[0094] Mutant PsiDKM colonies that were negative for ampicillin resistance and positive for kanamycin resistance were then inoculated in 2 mL of AMM with 150 mg/L 4-hydroxyindole and no antibiotic from a preculture containing kanamycin in a 48-well plate medium throughput assay. Cultures were induced with 1 mM isopropyl b-D-l-thiogalactopyranoside (IPTG) after 4 hours of growth. Metabolite concentrations were determined by HPLC analysis (Adams et ah, 2019) and optical density measurements were taken at 600 nm 48 hours after inoculation.

[0095] An analogous cloning procedure was followed for the norbaeocystin pathway; the norbaeocystin pathway fragment was reacted with Tn5-EZ transposase (Lucigen) and electroporated in the same manner described above. The screening conditions for the norbaeocystin pathway were the same as for the psilocybin pathway except 250 mg/L 4-hydroxyindole was used instead of 150 mg/L.

[0096] The resulting productive mutants from the first method (gel extraction) are shown in LIGs. 12A-12B. The second method (PCR purification) produced many productive mutants. A rescreening of select mutants is shown in LIG. 13.

Transcriptional Optimization

[0097] The plasmid pCas9CR4 was used to express the Cas9 protein for counterselection during promoter optimization and was electroporated into top producing mutants.

[0098] The plasmid pKDsgRNA-T7 was assembled using CPEC from PCR products. pKDsgRNA-pl5 was used as a template for the PCR with the primer pairs P12 + P22 and P13 + P23. The primers P12 and P13 both have 20bp long tails that are homologous to the T7 promoter. pKDsgRNA-T7 was electroporated into top producing mutants containing pCas9.

[0099] All promoter constructs were synthesized using PCR. Primers were annealed with 50 bp homology tails for integration using the l-red recombinase system. Pour mutant T7-lac promoters were selected for screening: H10, H9, G6, and C4. The promoters are expected to be orthogonal to the Cas9 targeted T7 consensus promoter to facilitate counterselection without causing a DSB in positive mutants (Cress et ah, 2016). The differences are highlighted in Table 1, shown below. [0100] Table 1 shows the sequences of all T7 mutant promoters used in this study.

[0101] The PAM Proximal Seed Nucleation Region (PPSNR) contains three base pairs as shown in Table 1. The Protospacer Adjacent Motif (PAM) site contains NGG in the consensus T7 as shown in Table 1. The base pair differences between the consensus T7 and the mutant promoters are shown in bold. It has been shown that 2-3 base pair mismatches in the PPSNR is sufficient to avoid counterselection (Cress et al., 2016). Furthermore, two of the promoters, H9 and H10, do not contain the PAM site, further reducing the counterselection ability of Consensus T7-targeted Cas9 in hosts with these mutant promoters.

Results

Screening of Mutants After Integration of T7 Controlled Pathway

[0102] After recovery following electroporation, 121 T7-PsiDKM mutants showed kanamycin resistance and did not contain the template plasmid pKET-T7-PsiDKM as determined by a lack of ampicillin resistance. Of the screened mutants, only three showed psilocybin production above the lower limit of detection. The psilocybin production of each as well as the T7-PsiDKM vector and the promoter optimized HlO-PsiDKM vector are reported in FIG. 12A. FIG. 12A shows the psilocybin titers from mutants containing Psi-DKM compared to the promoter optimized expression vector (H10 vector) and a vector containing the same promoter as the mutants (T7- vector). All mutants outperformed the unoptimized vector, however, it is believed that mutants capable of greater production can be generated. After recovery following electroporation, 72 T7- PsiDK mutants showed kanamycin resistance and did not contain the template plasmid pKET- T7-PsiDK as determined by a lack of ampicillin resistance. Of the screened mutants, only one showed norbaeocystin production above the lower limit of detection. The norbaeocystin production of the mutant as well as the T7-PsiDK vector and the promoter optimized C4-PsiDK vector are reported in FIG. 12B. FIG. 12B shows the norbaeocystin titer from the productive mutant containing Psi-DK compared to the promoter optimized expression vector (C4 vector) and a vector containing the same promoter as the mutants (T7-vector). The mutant, 1.2, appeared comparable to the optimized vector, and was selected for use in the transcriptional optimization protocol. However, a greater number of productive mutants was desired to enlarge the integration mutant library.

[0103] The norbaeocystin titers of selected integration mutants are reported in FIG. 13. The standard deviation of duplicate fermentations are represented by error bars. pETM6-SDM2X-C4- PsiDK (pNor labeled “C4 vector”) is the optimized expression vector, and pETM6-SDM2X-T7- PsiDK (labeled “T7 vector”) is the unoptimized expression vector containing the same T7 promoter as the integration mutants. Three of the mutants were selected for the in vivo transcriptional optimization protocol. Those mutants are shown with black bars. Mutant 1.2 (not shown here) was generated previously and was also selected for the in vivo transcriptional optimization.

Footprinting

[0104] The genomic loci of integration was determined by Inverse PCR (IPCR). A graphical representation of this process is shown in FIG. 11. The genome is extracted by GeneJet Genomic Extraction Kit. The isolated genome is digested in parallel with two restriction enzymes, Sail and Bcul. Following inactivation of the restriction enzymes, the digest products will be ligated at low concentrations to self-circularize. Two primers, PI and P30, located in the integrated construct facing outward were used to PCR amplify the genomic region directly adjacent to the integration construct. The PCR product was subject to Sanger Sequencing (Genewiz) using P36. The sequence obtained from sequencing was queried using NCBI’s blastn function. Integration location results for select mutants are presented in Table 2, below. [0105] Table 2. The Mutant ID is shown with the integration loci and loci characterization.

[0106] The Mutants 1.2, 2.7, 7.11, and 8.11 were used in the IPCR footprinting protocol. The locations of the integrations in mutants 2.7 and 8.11 were elucidated, shown in FIG. 15, however, the locations of the integrations of mutants 1.2 and 7.11 have yet to be found due to there being a Sail cute site in the genomic DNA too close to the construct to allow enough of the genome to be sequenced to be able to determine the integration location. To remedy this, the extracted genomic DNA will be digested with Xhol and the IPCR and sequencing process repeated. The IPCR footprinting protocol will also be applied to all Psi-DK mutants shown in FIG. 13 as well as a select group of Psi-DKM mutants yet to generated.

Example 3. Transcriptional Optimization

[0107] Four T7 -lac promoters of various strength, C4, G6, H9, and H10, were designed for use in the no-SC AR (Reisch & Prather, 2015) protocol to execute in vivo transcriptional optimization. These primer inserts have 50 base pair homology arms for integration. In addition to the mutant T7 promoters, constitutive and growth phase dependent promoters may be assessed for their potential use for production. These classes of promoters may be of more value than the inducible T7 mutants. The inducing agent IPTG is a major cost that may be prohibitive at scale, so a promoter system that does not rely on a small molecule inducer may be especially valuable.

[0108] The plasmid pKDsgRNA-T7 was assembled using CPEC from PCR products. pKDsgRNA-pl5 was used as a template for the PCR with the primer pairs P12 + P22 and P13 + P23. The primers P12 and P13 both have 20bp long tails that are homologous to the T7 promoter. [0109] Mutant T7 promoters with 50 bp homology arms were synthesized by PCR using self annealing primer pairs P14+P15, P16+P17, P18+P19 and P20+P21. The promoters were concentrated to >100 ng / pL and pooled to final concentration of 100 ng of each promoter/2.25 pL.

[0110] Mutants 1.9 and 1.2, harboring pCas9CR4 and pKDsgRNA-T7, were grown in 50 mL of LB with chloramphenicol and spectinomycin at 30C. Following induction of the lambda-red recombinase system with 50 mM L-arabinose final concentration for 15 minutes, the culture was made electrocompetent and 2.25 pL of the promoter pool was electroporated. Following outgrowth in SOC at 30°C for 90 minutes, the cultures were spread on plates containing spectinomycin, chloramphenicol, and anhydrotetracycline (aTc) as well as on plates containing spectinomycin and chloramphenicol. Colonies from the plates containing aTc were subjected to production screening as described previously. 22 promoter mutants from each parent mutant (1.2 and 1.9) were screened. The titers are reported in FIG. 14.

[0111] The production of the T7 mutants with and without the plasmids were roughly equivalent. This indicates that plasmid curing will not be a necessary step in the scaled-up production using these transcriptionally optimized strains. The production of the original mutants was altered by the introduction of mutant promoters; however, it is unclear if the more productive mutants underwent promoter swapping because their titers remained largely unchanged. To this end, selected mutants are subjected to rescreening with a higher concentration of the substrate 4-OH indole (from 250 mg/L to 300 mg/L) to explore the potential differences in the production of the transcriptionally altered mutants.

Example 4. Counterselection Validation

[0112] In order to validate the counterselection ability of Cas9 against mutant strains containing the consensus T7 -lac promoter, the T7-mutant strains containing pCas9CR4 and pKDsgRNA-T7 are plated on chloramphenicol/spectinomycin and chloramphenicol/spectinomycin/aTc after growing overnight to induce the counterselection system. The optical density of a preculture is used to estimate cell density. Appropriate dilutions are made to plate 500 colonies on both plate types. Plates containing aTc induce the counterselection system contained on pCas9CR4 and pKDsgRNA-T7 creating a DSB, causing cell death. A difference in colony count validates the counterselection ability of the Cas9 system. Additionally, the counterselection can be cross- validated by separately transforming wild type cells containing pKDsgRNA-T7 and pCas9CR4 with pETM6 plasmids (ampicillin resistance) containing each of the mutant T7 promoters. After induction with aTc in a culture containing chloramphenicol and spectinomycin, the cell broths can be diluted and spread on a plate containing ampicillin and a plate without ampicillin. A difference in colony count between the plates with and without ampicillin in the T7 promoter containing strain of at least two orders of magnitude would cross-validate the counterselection ability of the Cas9 system. A difference in colony count between the T7 strain and the mutant promoter containing strains would cross validate the orthogonality of the mutant promoters.

Example 5. In Vivo Transcriptional Optimization

[0113] Once the counterselection system is validated, the in vivo promoter optimization is executed using the no-SCAR method: mutants harboring both pKDsgRNA-T7 and pCas9CR4 are grown up until an ODeoo of 0.4-0.6 is achieved. Arabinose is added (final concentration 50 mM) to induce the l-red recombinase system, and the culture is allowed to grow for 20 minutes before being made electrocompetent. The induced electrocompetent cells are electroporated with 200 ng of insert DNA. Initially, all promoters are electroporated separately to validate their orthogonality to the T7. A negative control is also performed, consisting of the same induced electrocompetent mutants electroporated with no integration fragment to rescue the cells from Cas9 counterselection through a DSB to the genomic DNA. In parallel, an equimolar pool of promoters is electroporated to demonstrate a more generalizable system of library creation. After a two-hour outgrowth, cultures are spread on LB agar plates containing spectinomycin, chloramphenicol, and aTc. The resultant promoter exchanged mutants are screened in a medium throughput 48 well plate assay for psilocybin or norbaeocystin production. The initial screening is done in singlicate. Productive colonies will be subjected to colony PCR using a universal reverse primer that binds downstream of the promoter in the pathway and mutant promoter specific primers which bind to the mutant promoter in order to categorize the mutants and determine any bias in the promoter swapping. Productive colonies representative of each promoter are rescreened in triplicate alongside the already transcriptionally optimized control vectors: pETM6-SDM2x-H10-PsiDKM and pETM6-SDM2x-C4-PsiDK. The titers of this rescreening are recorded as well as any integration bias for mutant promoters. Example 6. Scaled-Up Fermentations

[0114] Once top mutants are identified, the fermentation reactions are scaled up in shake flasks to ensure consistent production. Without wishing to be bound by theory, it is assumed that production will be at least as consistent as the analogous vector-based expression system. Here, consistency is defined as the standard deviation of triplicate fermentations. After consistent production is verified (s_mutant £ s_vector), the fermentations are scaled up to a benchtop bioreactor. Without wishing to be bound by theory, it is assumed that the fermentation conditions previously optimized for the vector-based production are optimal for the chromosome-based production. AMM supplemented with 5 g/L methionine and 5 g/L serine is used for PsiDKM mutants, and AMM supplemented with 5 g/L serine is used for PsiDK mutants. The pH setpoint is 6.5 and is controlled by the addition of KOH cascaded from a pH probe. The DO setpoint is 20% and is controlled by agitation caused by a variable RPM Rushton style impeller cascaded from a DO probe. The temperature setpoint is 37 °C and is controlled by a heat jacket cascaded from a temperature probe.

[0115] The potential length of the productive fermentation times is assessed by monitoring the flux through the exogenous pathway by HPLC methods. The productive fermentation length, defined as the time that the relevant product (psilocybin or norbaeocystin) titers are increasing is recorded and compared to the analogous vector-based production strain.

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Table 3: Sequences

Table 4. Table of strains and relevant properties

[0146] 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 transposomes, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell.

2. The method of claim 1, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 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 psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 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.

4. The method of claim 1, wherein the psiM gene encodes a polypeptide comprising the amino acid sequence of 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.

5. 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.

6. The method of claim 1, wherein the prokaryotic cell is contacted with a transposome comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.

7. The method of claim 6, 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.

8. The method of claim 1, wherein the prokaryotic cell is contacted with a transposome comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

9. The method of claim 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 a transposome comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

11. The method of claim 10, 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.

12. The method of claim 1, wherein the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT).

13. The method of any one of claims 1-12, wherein the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine and combinations thereof.

14. The method of claim 13, wherein the supplement is fed continuously to the host cell.

15. The method of any one of claims 1-14, wherein the host cell is grown in an actively growing culture.

16. The method of any one of claims 1-15, further comprising use of transcriptional balancing on the prokaryotic host cell genome to enhance specifically psilocybin, an intermediate, or a side product thereof.

17. The method of claim 16, wherein the transcriptional balancing is achieved using a promoter 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.

18. The method of claim 16 or 17, wherein the transcriptional balancing is mediated through site specific Cas9 counterselection against an unoptimized construct.

19. A recombinant prokaryotic cell comprising one or more transposons, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

20. The recombinant prokaryotic cell of claim 19, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 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.

21. The recombinant prokaryotic cell of claim 19, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 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.

22. The recombinant prokaryotic cell of claim 19, wherein the psiM gene encodes a polypeptide comprising the amino acid sequence of 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.

23. The recombinant prokaryotic cell of claim 19, 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.

24. The recombinant prokaryotic cell of claim 19, wherein the transposon comprises a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.

25. The recombinant prokaryotic cell of claim 24, 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.

26. The recombinant prokaryotic cell of claim 19, wherein the transposon comprises a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

27. The recombinant prokaryotic cell of claim 26, 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.

28. The recombinant prokaryotic cell of claim 19, wherein the transposon comprises a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

29. The recombinant prokaryotic cell of claim 28, 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.

30. A transposome comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.

31. The transposome of claim 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. A transposome comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

33. The transposome of claim 32, 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.

34. A transposome comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

35. The transposome of claim 34, 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.

36. A transfection kit comprising a transposon comprising a psiD gene, a psiK gene, and a psiM gene all under control of a single promoter in operon configuration.

37. A transfection kit comprising a transposon comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

38. A transfection kit comprising a transposon comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

39. A method for the production of norbaeocystin comprising: contacting a prokaryotic host cell with one or more transposomes, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof; and culturing the host cell.

40. The method of claim 39, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 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.

41. The method of claim 39, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 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.

42. The method of claim 39, 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.

43. The method of claim 39, wherein the prokaryotic cell is contacted with a transposome comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, all under control of a single promoter in operon configuration.

44. The method of claim 43, 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.

45. The method of claim 39, wherein the prokaryotic cell is contacted with a transposome comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.

46. The method of claim 45, 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.

47. The method of claim 39, wherein the prokaryotic cell is contacted with a transposome comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in monocistronic configuration.

48. The method of claim 47, 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.

49. The method of any one of claims 39-48, wherein the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine and combinations thereof.

50. The method of claim 49, wherein the supplement is fed continuously to the host cell.

51. The method of any one of claims 39-50, wherein the host cell is grown in an actively growing culture.

52. A recombinant prokaryotic cell comprising one or more transposons, wherein each transposome comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof.

53. The recombinant prokaryotic cell of claim 52, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 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.

54. The recombinant prokaryotic cell of claim 52, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 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.

55. The recombinant prokaryotic cell of claim 52, 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.

56. The recombinant prokaryotic cell of claim 52, wherein the transposon comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, all under control of a single promoter in operon configuration.

57. The recombinant prokaryotic cell of claim 56, 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.

58. The recombinant prokaryotic cell of claim 52, wherein the transposon comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.

59. The recombinant prokaryotic cell of claim 58, 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.

60. The recombinant prokaryotic cell of claim 52, wherein the transposon comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.

61. The recombinant prokaryotic cell of claim 60, 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.

62. A transposome comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, all under control of a single promoter in operon configuration.

63. The transposome of claim 62, 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.

64. A transposome comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.

65. The transposome of claim 64, 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.

66. A transposome comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.

67. The transposome of claim 66, 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.

68. A transfection kit comprising a transposon comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, all under control of a single promoter in operon configuration.

69. A transfection kit comprising a transposon comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.

70. A transfection kit comprising a transposon comprising a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.