WO2022150854A1

WO2022150854A1 - Systems and methods for pharmaceutical production of psilocybin and intermediates or side products

Info

Publication number: WO2022150854A1
Application number: PCT/US2022/070132
Authority: WO
Inventors: John Andrew Jones; JR. William Jonathan GIBBONS
Original assignee: Miami University
Priority date: 2021-01-11
Filing date: 2022-01-11
Publication date: 2022-07-14
Also published as: WO2022150854A9

Abstract

Provided are systems and methods for the production of psilocybin or an intermediate or a side product thereof. In certain embodiments, the recombinant host strain employed is eukaryotic and is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803 In certain embodiments, the recombinant host strain employed is prokaryotic and 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. In some embodiments, the recombinant host strain is grown in an actively growing culture.

Description

SYSTEMS AND METHODS FOR PHARMACEUTICAL 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 systems and methods for pharmaceutical 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. 63/135,864, filed January 11, 2021, 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 January 9, 2022, is named 315691-00018_Sequence_Listing and is 49,108 bytes in size.

BACKGROUND

[0004] Research involving recombinant DNA began in the early 1970s as Paul Berg and coworkers created the first DNA molecule that contained genes from two different organisms. They inserted genes originating from the lambda bacteriophage and Escherichia coli (E. coli) into the DNA of simian virus 40 (SV40). Their work, published in 1972, provided the foundation for future recombinant DNA technology [1], Herbert Boyer and Stanley Cohen expanded upon this work in 1973 when they published their discovery that genetically engineered plasmids containing recombinant genes can retain function and stably replicate across multiple generations inside an E. coli host organism [2].

[0005] This early success was quickly followed by concerns from scientists involving the safety of recombinant DNA, leading to the Asilomar Conference on Recombinant DNA Molecules in 1975 [3,4]. Based upon the summary statement from the conference, the National Institutes of Health (NIH) created a document entitled, “Guidelines for Research Involving Recombinant DNA Molecules,” which has been updated many times and retitled since its release [5,6].

[0006] Recombinant DNA research continued with caution and new scientific techniques emerged that facilitated its widespread adoption. Restriction enzyme digestion, which cuts DNA in a site- specific manner based upon the DNA recognition sequence of the restriction enzyme, and DNA ligation, which connects two DNA fragments having complementary overhangs, are two such enabling technologies [7-9]. These techniques, along with many others, have made recombinant DNA technology a staple molecular biology tool [2]. More recently, techniques such as CRISPR technology for genome editing have added functionality to the recombinant DNA toolbox [10]. Genetic engineering and biomanufacturing technology have led to many advancements in medicine [11,12], agriculture [13,14], and energy [15,16] and continue to be an enabling technology for cutting edge discoveries [17]. Despite the widespread impact of recombinant DNA technologies, public concern and controversy still remain [18-22].

[0007] Recently, recombinant DNA has been used to produce psilocybin, the chemical found in psychedelic mushrooms that causes a hallucinogenic response upon ingestion. This method for producing psilocybin became possible after the Hoffmeister group reported the metabolic pathway for the biosynthetic production of psilocybin [23].

[0008] Natural sources of psilocybin such as Psilocybe sp. and related mushrooms have been historically consumed by some native populations for religious and ceremonial purposes [24- 26]. In modern times, psilocybin has been popularized by the recreational use of these so called ‘magic’ mushrooms, and the resulting psychedelic effects they have on the user. Psilocybin has returned to the spotlight due to recent positive results from clinical trials for the treatment of a variety of neurological issues, including treatment-resistant depression, post-traumatic stress disorder (PTSD), cancer related anxiety, substance abuse, and more [27-30].

[0009] There remains a need for systems and methods for pharmaceutical production of psilocybin and intermediates or side products thereof. SUMMARY

[0010] Provided is a system configured to produce psilocybin or an intermediate or a side product thereof, comprising: a culture comprising a recombinant prokaryotic or eukaryotic host strain; a suspension mechanism configured to suspend the culture; a water bath; a temperature control device set at a temperature of about 4 °C to about 40 °C, optionally inserted in the water bath; and one or more containers configured to hold and grow the culture to produce psilocybin or an intermediate or a side product thereof, and placed in the water bath.

[0011] In some embodiments, the system further comprises at least one pump connected to tubing that is placed in each of the one or more containers and optionally an air diffusion device connected to the tubing and placed in the culture.

[0012] In some embodiments, the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 8-16 hours, for example overnight. In further embodiments, the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 12 hours.

[0013] In some embodiments, the recombinant host strain is eukaryotic. In some embodiments, the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

[0014] In some embodiments, the recombinant host strain is prokaryotic. In some embodiments, the recombinant host strain 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, 4-hydroxyindole is added to the one or more containers.

[0016] In some embodiments, an antibiotic, for example ampicillin, is added to the culture to select for the recombinant prokaryotic host strain and limit contamination.

[0017] In some embodiments, the at least one pump comprises an aquarium pump.

[0018] In some embodiments, the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

[0019] In further embodiments, 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.

[0020] In further embodiments, 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.

[0021] In further embodiments, 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.

[0022] In some embodiments, the recombinant host strain comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In further 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.

[0023] In some embodiments, the recombinant host strain is contacted with an expression vector 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 further 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] 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).

[0025] In some embodiments, the intermediate of psilocybin is norbaeocystin and wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof.

[0026] In some embodiments, the recombinant host strain 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 recombinant host strain.

[0027] In some embodiments, the recombinant host strain is grown in an actively growing culture.

[0028] Also provided is a method for producing psilocybin or an intermediate or a side product thereof, comprising: culturing a recombinant prokaryotic or eukaryotic host strain in one or more containers; placing the one or more containers in a water bath; inserting a temperature control device, set at a temperature of about 4 °C to about 40 °C, in the water bath; placing the water bath on or below a suspension mechanism; and growing the psilocybin or an intermediate or a side product thereof in the one or more containers. [0029] In some embodiments, the method further comprises providing at least one pump connected to tubing that is placed in each of the one or more containers and optionally providing an air diffusion device connected to the tubing and placed in the culture.

[0030] In some embodiments, the recombinant host strain was inoculated from a culture that was grown at least 8-16 hours, for example overnight. In further embodiments, the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 12 hours.

[0031] In some embodiments, the recombinant host strain is eukaryotic. In some embodiments, the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

[0032] In some embodiments, the recombinant host strain is prokaryotic. In some embodiments, the recombinant host strain 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.

[0033] In some embodiments, 4-hydroxyindole is added to the one or more containers.

[0034] In some embodiments, an antibiotic, for example ampicillin, is added to the culture to select for the recombinant prokaryotic host strain and limit contamination.

[0035] In some embodiments, the at least one pump comprises an aquarium pump.

[0036] In some embodiments, the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

[0037] In further embodiments, 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.

[0038] In further embodiments, 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.

[0039] In further embodiments, 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.

[0040] In some embodiments, the recombinant host strain comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In further 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.

[0041] In some embodiments, the recombinant host strain is contacted with an expression vector 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 further 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.

[0042] 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).

[0043] In some embodiments, the intermediate of psilocybin is norbaeocystin and wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof. [0044] In some embodiments, the recombinant host strain 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 recombinant host strain.

[0045] In some embodiments, the recombinant host strain is grown in an actively growing culture.

[0046] Also provided is a kit comprising the system of any of the embodiments described herein.

[0047] Also provided is a psilocybin or an intermediate or a side product thereof compound prepared by employing the system of any of the embodiments described herein.

[0048] Provided is a psilocybin or an intermediate or a side product thereof compound prepared by the method of any of the embodiments described herein.

[0049] Provided is a psilocybin or an intermediate or a side product thereof composition comprising a psilocybin or an intermediate or a side product thereof compound, respectively, prepared by employing the system of any of the embodiments described herein, and a carrier and/or an excipient.

[0050] Also provided is a psilocybin or an intermediate or a side product thereof composition comprising a psilocybin or an intermediate or a side product thereof compound, respectively, prepared by the method of any of the embodiments described herein, and a carrier and/or an excipient.

DESCRIPTION OF THE FIGURES

[0051] The foregoing summary, as well as the following detailed description of the presently disclosed technology, will be better understood when read in conjunction with the appended drawings, wherein like numerals designate like elements throughout. For the purpose of illustrating the presently disclosed technology, there are shown in the drawings various illustrative embodiments. It should be understood, however, that the presently disclosed technology is not limited to the precise arrangements and instrumentalities shown. In the drawings: [0052] FIG. 1 shows a visual representation of the homebrew setup according to one embodiment of the present technology.

[0053] FIG. 2 shows psilocybin titers under each set of culture conditions of the present technology, wherein the star symbol (*) denotes significant difference, p < 0.01 and the initials “n.s.” denotes no significant difference, p > 0.1.

[0054] FIG. 3 shows time course profiles for the final product norbaeocystin, key intermediate 4- hydroxytryptophan, and substrate 4-hydroxyindole of the present technology.

[0055] FIG. 4 shows a UV absorbance (280 nm) chromatogram according to the present technology.

[0056] FIG. 5 shows MS extracted ion chromatograph at [M-H]⁺ = 257 m/z as expected for norbaeocystin according to the present technology.

[0057] FIG. 6 shows a mass spectrum of the peak identified as norbaeocystin according to the present technology.

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

[0063] As used herein, the term “prokaryotic host cell” means a prokaryotic cell that is susceptible to transformation, transfection, transduction, or the like, with a nucleic acid construct or expression vector comprising a polynucleotide. The term “prokaryotic host cell” encompasses any progeny that is not identical due to mutations that occur during replication.

[0064] 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.

[0065] 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.

[0066] 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).

[0067] As used herein, the term “suspension mechanism” means a stir plate with stir bar, a significant flow of air, an overhead mixer, or anything else that is configured to keep a culture in suspension.

[0068] As used herein, the term “air diffusion device” means air stones, a bubbler, an air filtration system, or anything else that is configured to assist with air diffusion. [0069] The systems, 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.

[0070] In some embodiments of any of the systems, materials, methods or compositions described herein, a range is intended to comprise every integer or fraction or value within the range.

Systems, methods, and kits for the production of psilocybin or an intermediate or a side product thereof

Systems

[0071] Provided is a system for producing psilocybin or an intermediate or a side product thereof, comprising: a culture (1) comprising a recombinant prokaryotic or eukaryotic host strain; a suspension mechanism (2) configured to suspend the culture; a water bath (3); a temperature control device (4) set at a temperature of about 4 °C to about 40 °C, optionally inserted in the water bath; and one or more containers (5) configured to hold and grow the culture to produce psilocybin or an intermediate or a side product thereof, and placed in the water bath (3).

[0072] In some embodiments, the system further comprises at least one pump (6) (shown schematically in FIG. 1) connected to tubing (7) that is placed in each of the one or more containers (5) and optionally an air diffusion device (8) connected to the tubing (7) and placed in the culture (1).

[0073] In some embodiments, two 500 mL bottles, containing inoculated cultures, magnetic stir bars, and air stones, are placed into a plastic tub filled with water. The air stones are attached to small aquarium air pumps via plastic tubing. The tops of the bottles may be covered with aluminum foil. The tub is placed onto two magnetic stir plates, set to 510 RPM. A sous vide, set to 37 °C, may be attached to the plastic tub and inserted into the water bath.

[0074] In some embodiments, the temperature control device is set at a temperature of about 10 °C to about 40 °C. In further embodiments, the temperature control device is set at a temperature of about 20 °C to about 40 °C. In further embodiments, the temperature control device is set at a temperature of about 30 °C to about 40 °C. In yet further embodiments, the temperature control device is set at a temperature of about 30 °C, 31 °C, 32 °C, 33 °C, 34°C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C, for example about 37 °C.

[0075] In some embodiments, the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 8-16 hours, for example overnight. In further embodiments, the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 12 hours.

[0076] In some embodiments, the recombinant host strain is eukaryotic. In some embodiments, the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

[0077] In some embodiments, the recombinant host strain is prokaryotic. In some embodiments, the recombinant host strain 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.

[0078] In some embodiments, 4-hydroxyindole is added to the one or more containers.

[0079] In some embodiments, an antibiotic, for example ampicillin, is added to the culture to select for the recombinant prokaryotic host strain and limit contamination.

[0080] In some embodiments, the at least one pump comprises an aquarium pump. [0081] In some embodiments, the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

[0082] In certain embodiments, 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. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0083] In certain embodiments, 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. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 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 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. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 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.

[0085] In some embodiments, the recombinant host strain comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In further 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.

[0086] In some embodiments, the recombinant host strain is contacted with an expression vector 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 further 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.

[0087] Any configuration or arrangement of promoters and terminators is envisaged.

[0088] It is envisaged that any intermediate or side product of psilocybin may be produced by any of the systems described herein. In some embodiments, the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aemginascin, 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 aemginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4- OH-TMT).

[0089] In some embodiments, the intermediate of psilocybin is norbaeocystin and wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof. [0090] In certain embodiments, 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. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0091] In certain embodiments, the psiK comprises 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. In certain embodiments, the psiK gene comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 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.

[0092] In some embodiments, the recombinant host strain is eukaryotic. In some embodiments, the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

[0093] In some embodiments, the recombinant host strain is prokaryotic. In some embodiments, the recombinant host strain 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. [0094] In certain embodiments, the recombinant host strain is contacted with an expression vector comprising a psilocybin production gene selected from the group consisting of a psiD gene, a psiK gene, and combinations thereof, all under control of a single promoter in operon configuration. In certain embodiments, the recombinant host strain is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged. In certain embodiments, none of the expression vectors comprises a psiM gene.

[0095] 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.

[0096] In certain embodiments, the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4-hydroxytryptophan, 4-hydroxytryptamine, and combinations thereof. In certain exemplary embodiments, the supplement is fed continuously to the recombinant host strain. In further embodiments, the recombinant host strain is grown in an actively growing culture.

Psilocybin

[0097] The psilocybin and intermediate or side products are found extracellularly in the fermentation broth. In certain embodiments, the psilocybin and intermediate or side products are isolated. These target products can be collected through drying the fermentation broth after centrifugation to remove the cell biomass. The resulting dry product can be extracted to further purify the target compounds. Alternatively, the products can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions psilocybin or any of the intermediate or side products into the organic phase. Furthermore, contaminants from the fermentation broth can be removed through extraction leaving the psilocybin and/or intermediate or side products in the aqueous phase for collection after drying or crystallization procedures. [0098] In certain embodiments, the systems described herein result in a titer of psilocybin of about 0.01 to about 50 g/L. In some embodiments, the systems described herein result in a titer of psilocybin of about 0.01 to about 10 g/L. In yet further embodiments, the systems described herein result in a titer of psilocybin of about 0.01 to about 2 g/L. In certain embodiments, the systems described herein result in a titer of psilocybin of about 0.1 to about 1.0 g/L. In further embodiments, the systems described herein result in a titer of psilocybin of about 0.3 g/L.

[0099] In certain embodiments, the systems described herein result in a molar yield of psilocybin of about 10% to about 100%. In some embodiments, the systems described herein result in a molar yield of psilocybin of about 20% to about 80%. In yet further embodiments, the systems described herein result in a molar yield of psilocybin of about 30% to about 70%. In certain embodiments, the systems described herein result in a molar yield of psilocybin of about 30% to about 60%. In further embodiments, the systems described herein result in a molar yield of psilocybin of about 40%.

Norbaeocystin

[0100] The norbaeocystin is found extracellularly in the fermentation broth. In certain embodiments, the norbaeocystin is isolated. Norbaeocystin 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 norbaeocystin. Alternatively, the norbaeocystin can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions norbaeocystin into the organic phase. Furthermore, contaminants from the fermentation broth can be removed through extraction leaving the norbaeocystin in the aqueous phase for collection after drying or crystallization procedures.

[0101] In certain embodiments, the methods described herein result in a titer of norbaeocystin of about 0.01 to about 50 g/L. In some embodiments, the systems described herein result in a titer of norbaeocystin of about 0.01 to about 10 g/L. In yet further embodiments, the systems described herein result in a titer of norbaeocystin of about 0.01 to about 2 g/L. In certain embodiments, the systems described herein result in a titer of norbaeocystin of about 0.1 to about 1.0 g/L. In further embodiments, the systems described herein result in a titer of norbaeocystin of about 0.3 g/L.

[0102] In certain embodiments, the methods described herein result in a molar yield of norbaeocystin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of norbaeocystin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of norbaeocystin of about 30% to about 60%. In further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 40%.

Methods

[0103] Provided is a method for producing psilocybin or an intermediate or a side product thereof, comprising: culturing a recombinant prokaryotic or eukaryotic host strain in one or more containers; placing the one or more containers in a water bath; inserting a temperature control device, set at a temperature of about 4 °C to about 40 °C, in the water bath; placing the water bath on or below a suspension mechanism; and growing the psilocybin or an intermediate or a side product thereof in the one or more containers.

[0104] In some embodiments, the method further comprises providing at least one pump connected to tubing that is placed in each of the one or more containers and optionally providing an air diffusion device connected to the tubing and placed in the culture.

[0105] In some embodiments, the temperature control device is set at a temperature of about 10 °C to about 40 °C. In further embodiments, the temperature control device is set at a temperature of about 20 °C to about 40 °C. In further embodiments, the temperature control device is set at a temperature of about 30 °C to about 40 °C. In yet further embodiments, the temperature control device is set at a temperature of about 30 °C, 31 °C, 32 °C, 33 °C, 34°C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C, for example about 37 °C.

[0106] In some embodiments, the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 8-16 hours, for example overnight. In further embodiments, the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 12 hours.

[0107] In some embodiments, the recombinant host strain is eukaryotic. In some embodiments, the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

[0108] In some embodiments, the recombinant host strain is prokaryotic. In some embodiments, the recombinant host strain 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.

[0109] In some embodiments, 4-hydroxyindole is added to the one or more containers.

[0110] In some embodiments, an antibiotic, for example ampicillin, is added to the culture to select for the recombinant prokaryotic host strain and limit contamination.

[0111] In some embodiments, the at least one pump comprises an aquarium pump.

[0112] In some embodiments, the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

[0113] In certain embodiments, 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. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0114] In certain embodiments, 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. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 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.

[0115] In certain embodiments, 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. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 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.

[0116] In some embodiments, the recombinant host strain comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In further 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.

[0117] In some embodiments, the recombinant host strain is contacted with an expression vector 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 further 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.

[0118] Any configuration or arrangement of promoters and terminators is envisaged.

[0119] It is envisaged that any intermediate or side product of psilocybin may be produced by any of the methods described herein. In some embodiments, the intermediate or side product of psilocybin is 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).

[0120] In some embodiments, the intermediate of psilocybin is norbaeocystin and wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof.

[0121] In certain embodiments, 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. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0122] In certain embodiments, 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. In certain embodiments, the psiK gene comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2or 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.

[0123] In some embodiments, the recombinant host strain is eukaryotic. In some embodiments, the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

[0124] In some embodiments, the recombinant host strain is prokaryotic. In some embodiments, the recombinant host strain 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.

[0125] In certain embodiments, the recombinant host strain is contacted with an expression vector comprising a psilocybin production gene selected from the group consisting of a psiD gene, a psiK gene, and combinations thereof, all under control of a single promoter in operon configuration. In certain embodiments, the recombinant host strain is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged. In certain embodiments, none of the expression vectors comprises a psiM gene.

[0126] 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.

[0127] In certain embodiments, the recombinant host strain is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4- hydroxytryptophan, 4-hydroxytryptamine, and combinations thereof. In certain exemplary embodiments, the supplement is fed continuously to the recombinant host strain. In further embodiments, the recombinant host strain is grown in an actively growing culture.

Psilocybin

[0128] The psilocybin and intermediate or side products are found extracellularly in the fermentation broth. In certain embodiments, the psilocybin and intermediate or side products are isolated. These target products can be collected through drying the fermentation broth after centrifugation to remove the cell biomass. The resulting dry product can be extracted to further purify the target compounds. Alternatively, the products can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions psilocybin or any of the intermediate or side products into the organic phase. Furthermore, contaminants from the fermentation broth can be removed through extraction leaving the psilocybin and/or intermediate or side products in the aqueous phase for collection after drying or crystallization procedures.

[0129] In certain embodiments, the methods described herein result in a titer of psilocybin of about 0.01 to about 50 g/L. In some embodiments, the methods described herein result in a titer of psilocybin of about 0.01 to about 10 g/L. In yet further embodiments, the methods described herein result in a titer of psilocybin of about 0.01 to about 2 g/L. In certain embodiments, the methods described herein result in a titer of psilocybin of about 0.1 to about 1.0 g/L. In further embodiments, the methods described herein result in a titer of psilocybin of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 g/L. [0130] In certain embodiments, the methods described herein result in a molar yield of psilocybin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of psilocybin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of psilocybin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of psilocybin of about 30% to about 60%. In further embodiments, the methods described herein result in a molar yield of psilocybin of about 40%.

Norbaeocystin

[0131] The norbaeocystin is found extracellularly in the fermentation broth. In certain embodiments, the norbaeocystin is isolated. Norbaeocystin 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 norbaeocystin. Alternatively, the norbaeocystin can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions norbaeocystin into the organic phase. Furthermore, contaminants from the fermentation broth can be removed through extraction leaving the norbaeocystin in the aqueous phase for collection after drying or crystallization procedures.

[0132] In certain embodiments, the methods described herein result in a titer of norbaeocystin of about 0.01 to about 50 g/L. In some embodiments, the methods described herein result in a titer of norbaeocystin of about 0.01 to about 10 g/L. In yet further embodiments, the methods described herein result in a titer of norbaeocystin of about 0.01 to about 2 g/L. In certain embodiments, the methods described herein result in a titer of norbaeocystin of about 0.1 to about 1.0 g/L. In further embodiments, the methods described herein result in a titer of psilocybin of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 g/L.

[0133] In certain embodiments, the methods described herein result in a molar yield of norbaeocystin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of norbaeocystin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of norbaeocystin of about 30% to about 60%. In further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 40%.

Kits, compounds and compositions

[0134] Also provided is a kit comprising the system of any of the embodiments described herein.

[0135] Also provided is a psilocybin or an intermediate or a side product thereof compound prepared by employing the system of any of the embodiments described herein.

[0136] Provided is a psilocybin or an intermediate or a side product thereof compound prepared by the method of any of the embodiments described herein.

[0137] Provided is a psilocybin or an intermediate or a side product thereof composition comprising a psilocybin or an intermediate or a side product thereof compound, respectively, prepared by employing the system of any of the embodiments described herein, and a carrier and/or an excipient.

[0138] Also provided is a psilocybin or an intermediate or a side product thereof composition comprising a psilocybin or an intermediate or a side product thereof compound, respectively, prepared by the method of any of the embodiments described herein, and a carrier and/or an excipient.

Composition

[0139] In some embodiments, the composition is a parenteral dosage form. In some embodiments, the composition is an oral dosage form. In some embodiments, the composition comprises a tablet, capsule, dry powder, gel, film, solution or combination.

Carriers

[0140] The compositions described herein may comprise a carrier. In some embodiments, the carrier is a solvent (e.g. an alcohol), a polymer, a nanoparticle, a liposome, a lipoprotein, a gel, a sugar or sugars, protein or other matrix, or carriage device. Route of Delivery

[0141] In some embodiments, the route of delivery is by injection, oral, sublingual, buccal, transdermal, or nasal.

EXAMPLES

Example 1

Materials and Methods

Bacterial strains, vectors, and media

[0142] E. coli BL21 star™ (DE3), containing the psilocybin pathway expression plasmid pPsilol6 (described in PCX Patent Application PCT/US2020/051543, published as WO202 1/086513, which is hereby incorporated by reference in its entirety and for all purposes), was used for all production cultures [37]. Modified Andrew’s Magic Media (MAMM) was used for all cultivation conditions [46]. Production cultures were induced with 1 mM isopropyl b-D-l- thiogalactopyranoside (IPTG) four hours after inoculation with an overnight culture.

[0143] The MAMM used in this study consisted of 3.5 g/L KH₂PO₄, 5.0 g/L K₂HPO₄, 3.5 g/L (NH₄)₂HRO₄, 2 g/L casamino acids, 100 mL of lOx Modified Mix, 1 mL of 1 M MgSCL, 1 mL of 100 mM CaCh, 1 mL of 1.0 g/L thiamine HC1, 40 g/L glucose, 5 g/L of serine, 5 g/L methionine, and 150 mg/L of 4-hydroxyindole. The lOx Modified Mix consisted of 28 mg/L LeSCL VthO, 29.2 g/L NaCl, 5.1 g/L NH₄C1, 1.1 g/L MgCl₂, 0.48 g/L K₂SO₄, 0.2 mL Micronutrient Stock. The micronutrient stock consisted of 0.18 g/L (NH4)_όMq7q24, 1.24 g/L H₃BO₃, 0.12 g/L CuSCL,

0.8 g/L MnCl₂, 0.14 g/L ZnSO_4.

[0144] The homebrew fermentations were additionally supplemented with a carbon and nitrogen feed solution (500 g/L glucose and 90 g/L (NLL₄)₂HPCL) and substrate feed solution (40 mg/mL 4-hydroxyindole). Live milliliters of the carbon and nitrogen feed solution was supplemented to the 250 mL working volume fermentation at approximately 8 and 32 hours after inoculation. An appropriate volume of the substrate feed solution was supplemented to the fermentation to result in an addition of 150 mg/L of 4-hydroxyindole approximately 8 and 32 hours after inoculation. Results

[0145] A rudimentary setup was constructed using low-cost and easily available items (FIG. 1). Water bath temperature was controlled at 37 °C using a residential grade sous vide. The parallel cultures were aerated using small aquarium air pumps with attached air stones. The stir rate was set to 510 RPM on a magnetic stir plate. All cultures were grown in 250 mL of MAMM in 500 mL glass bottles. Boluses of 4-hydroxyindole (150 mg/L final concentration) were added initially and as needed to maintain pathway flux towards psilocybin. Psilocybin production was quantified via HPLC using methods described previously [37]. Samples were prepared by centrifugation at 12000 x g for 5 minutes. 2 pL of the resulting supernatant was then injected for HPLC or LC-MS analysis. Analysis was performed on a Thermo Scientific Ultimate 3000 High- Performance Liquid Chromatography (HPLC) system equipped with Diode Array Detector (DAD) and Refractive Index Detector (RID). Authentic standards were purchased for glucose (Sigma), psilocybin (Cerilliant), and 4-hydroxyindole (BioSynth). Standards for baeocystin, norbaeocystin, 4-hydroxytryptamine, and 4-hydroxytryptophan were quantified using a standard for a similar analog due to limited commercial availability. Norbaeocystin was quantified using a standard purified in house via preparative HPLC. 4-hydroxytryptamine and 4-hydroxytryptophan were quantified on the standard curves of 5-hydroxytryptamine (Alfa Aesar) and 5- hydroxytryptophan (Alfa Aesar), respectively. No significant intracellular accumulation of target metabolites was observed upon analysis with and without cell lysis. Without wishing to be bound by theory, transport across the cell membrane was assumed to be passive. Glucose analysis was performed using an Aminex HPX-87H column maintained at 30 °C followed by a refractive index detector (RID) held at 35 °C. The mobile phase was 5 mM H2SO4 in water at a flow rate of 0.6 mL/min. Glucose was quantified using a standard curve with a retention time of 8.8 min.

[0146] UV absorbance at 280 nm was used to quantify all aromatic compounds. Analysis was performed using an Agilent ZORBAX Eclipse XDB-C18 analytical column (3.0 mm x 250 mm,

5 mhi) with mobile phases of acetonitrile (A) and water (B) both containing 0.1% formic acid at a flow rate of 1 mL/min: 0 min, 5% A; 0.43 min, 5% A; 5.15 min, 19% A; 6.44 min, 100 % A;

7.73 min 100% A; 7.73 min, 5% A; 9.87 min, 5% A. This method resulted in the following observed retention times: psilocybin (2.2 min), baeocystin (1.7 min), norbaeocystin (1.9 min), 4- hydroxytryptamine (3.4 min), 4-hydroxytryptophan (3.6 min), and 4-hydroxyindole (6.6 min).

[0147] Three sets of experimental conditions were tested using six replicates each. First, the cultures were grown using standard sterile technique; all glassware was autoclaved, the media was filter sterilized under flame, all samples were extracted under flame, and ampicillin was used to select for the recombinant strain and limit contamination, referred to here as ‘Standard Conditions’. Next, the glassware was ethanol rinsed, the media was not filter sterilized, samples were extracted without a flame, and ampicillin was not used, referred to here as ‘Basic Homebrew Conditions’. Third, the same setup as Basic Homebrew Conditions was used, except ampicillin was present, referred to here as ‘Homebrew with Ampicillin Conditions.’

[0148] Standard Conditions produced the highest average titer at 366 ± 67 mg/L of psilocybin, followed by the Homebrew with Ampicillin Conditions producing an average titer of 319 ± 27 mg/L of psilocybin, while the Basic Homebrew Conditions produced the lowest average titer out of the conditions tested at 247 ± 34 mg/L of psilocybin (FIG. 2). The addition of ampicillin to the Basic Homebrew Conditions improved titers significantly (p < 0.01), presumedly due to better plasmid stability and less microbial competition under ampicillin selection. Several other trials, with varied media compositions and conditions, produced even higher titers of psilocybin, thus demonstrating that yields could be increased with further optimization. These conditions were not pursued due to the higher level of process complexity, which was not appropriate for our simple homebrew environment simulation. Raw data and the results of statistical tests of significance can be found in Tables 1-4 below.

Table 1. Results from homebrew conditions evaluation studies (N=6).

Table 2. Two-tailed, unpaired t-Test Result comparing Standard and Homebrew conditions.

Table 3. Two-tailed, unpaired t-Test Result comparing Standard and Ampicillin conditions.

Table 4. Two-tailed, unpaired t-Test Result comparing Homebrew and Ampicillin conditions.

Example 2: Production of Norbaeocvstin

[0149] The examples demonstrated in this work can directly be utilized to produce norbaeocvstin and other tryptamine derivatives with only minor modification. For the case of norbaeocystin, the genetically optimized E. coli strain, BL21 star^lM (DE3) containing the plasmid pETM6-C4- psiD-psiK (operon configuration) as described in PCT Patent Application (PCT/US2020/051543, published as WO2021/086513, which is hereby incorporated by reference in its entirety and for all purposes) performs the biocatalysis, and the methionine is removed from the media composition, resulting in an even lower cost production process. All other process details remain the same, highlighting the broad applicability of this process. Example 3: Enhanced Production of Norbaeocvstin

Materials and Methods

Bacterial strains, vectors, and media

[0150] BL21 star™ (DE3) containing the pNor plasmid was employed for production of norbaeocystin. pNor is the basic operon configuration of psiD and psiK under the control of the strong C4 mutant T7 promoter located on the pETM6-SDM2x expression vector.

Results

[0151] The sole variation from Example 1 is in the base media composition, where in the case of norbaeocystin production, the methionine supplement was omitted. Without wishing to be bound by theory, enhanced levels of the S-adenosyl methionine methyl donor are not necessary for norbaeocystin production. The time course profiles for the final product norbaeocystin, key intermediate 4-hydroxytryptophan, and substrate 4-hydroxyindole are shown in FIG. 3. Here may be seen a total of three 4-hydroxyindole additions (150 mg/L each) at timepoints 0, 7.75, and 22.75 hours post inoculation. Overall norbaeocystin production is similar to that of our psilocybin case study, reaching a level of 318 +/- 9.3 mg/L in just under 48 hours (FIG. 3).

[0152] To confirm the identity of norbaeocystin, HPLC-MS analysis was performed on the fermentation samples. A UV absorbance (280 nm) chromatogram is presented in FIG. 4, with norbaeocystin eluting at approximately 1.6 minutes. A MS extracted ion chromatograph at the [M-H]⁺ = 257 m/z that was expected for norbaeocystin is shown in FIG. 5. The retention time is consistent with the UV trace in FIG. 4. Finally, the mass spectrum of the peak identified as norbaeocystin is shown in FIG. 6. The dominant mass is 257 m/z, which is in alignment with the expected m/z in positive ion mode.

[0153] References

1. Jackson DA, Symons RH, Berg P: Biochemical Method for Inserting New Genetic

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Table 5: Sequences

[0154] 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 system configured to produce psilocybin or an intermediate or a side product thereof, comprising: a culture comprising a recombinant prokaryotic or eukaryotic host strain; a suspension mechanism configured to suspend the culture; a water bath; a temperature control device set at a temperature of about 4 °C to about 40 °C, optionally inserted in the water bath; and one or more containers configured to hold and grow the culture to produce psilocybin or an intermediate or a side product thereof, and placed in the water bath.

2. The system of claim 1, further comprising at least one pump connected to tubing that is placed in each of the one or more containers and optionally an air diffusion device connected to the tubing and placed in the culture.

3. The system of claim 1, wherein the culture comprising the recombinant host strain was inoculated from a culture that was grown for at least 8-16 hours, for example overnight.

4. The system of claim 1, wherein the recombinant host strain is eukaryotic.

5. The system of claim 4, wherein the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

6. The system of claim 1, wherein the recombinant host strain is prokaryotic.

7. The system of claim 6, wherein the recombinant host strain 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.

8. The system of any one of claims 1-7, wherein 4-hydroxyindole is added to the one or more containers.

9. The system of claim 6 or 7, wherein an antibiotic, for example ampicillin, is added to the culture to select for the recombinant prokaryotic host strain and limit contamination.

10. The system of claim 1, wherein the at least one pump comprises an aquarium pump.

11. The system of any one of claims 1-10, wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

12. The system of claim 11, 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.

13. The system of claim 11, 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.

14. The system of claim 11, 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.

15. The system of claim 11, wherein the recombinant host strain comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.

16. The system of claim 15, 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.

17. The system of claim 11, wherein the recombinant host strain is contacted with an expression vector 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.

18. The system of claim 17, 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.

19. The system 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).

20. The system of any one of claims 1-10, wherein the intermediate of psilocybin is norbaeocystin and wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof.

21. The system of any one of claims 1-20, wherein the recombinant host strain is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine and combinations thereof.

22. The system of claim 21, wherein the supplement is fed continuously to the recombinant host strain.

23. The system of any one of claims 1-22, wherein the recombinant host strain is grown in an actively growing culture.

24. A method for producing psilocybin or an intermediate or a side product thereof, comprising: culturing a recombinant prokaryotic or eukaryotic host strain in one or more containers; placing the one or more containers in a water bath; inserting a temperature control device, set at a temperature of about 4 °C to about 40 °C, in the water bath; placing the water bath on or below a suspension mechanism; and growing the psilocybin or an intermediate or a side product thereof in the one or more containers.

25. The method of claim 24, further comprising providing at least one pump connected to tubing that is placed in each of the one or more containers and optionally providing an air diffusion device connected to the tubing and placed in the culture.

26. The method of claim 24, wherein the recombinant host strain was inoculated from a culture that was grown for at least 8-16 hours, for example overnight.

27. The method of claim 24, wherein the recombinant host strain is eukaryotic.

28. The system of claim 27, wherein the recombinant host strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces pastorianus, Brettanomyces, Aspergillus niger, Aspergillus nidulans, Chinese Hamster Ovary (CHO), Yarrowia lipolytica, Pichia pastoris, Synechococcus elongatus, and Synechocystis sp. PCC6803.

29. The method of claim 24, wherein the recombinant host strain is prokaryotic.

30. The method of claim 29, wherein the recombinant host strain 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.

31. The method of any one of claims 24-30, wherein 4-hydroxyindole is added to the one or more containers.

32. The method of claim 29 or 30, wherein an antibiotic, for example ampicillin, is added to the culture to select for the recombinant prokaryotic host strain and limit contamination.

33. The method of claim 24, wherein the at least one pump comprises an aquarium pump.

34. The method of any one of claims 24-33, wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.

35. The method of claim 34, 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.

36. The method of claim 34, 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.

37. The method of claim 34, 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.

38. The method of claim 34, wherein the recombinant host strain comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.

39. The method of claim 38, 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.

40. The method of claim 34, wherein the recombinant host strain is contacted with an expression vector 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.

41. The method of claim 40, 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.

42. The method of claim 24, 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).

43. The system of any one of claims 24-33, wherein the intermediate of psilocybin is norbaeocystin and wherein the recombinant host strain comprises one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof.

44. The method of any one of claims 24-43, wherein the recombinant host strain is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine and combinations thereof.

45. The method of claim 44, wherein the supplement is fed continuously to the recombinant host strain.

46. The method of any one of claims 24-45, wherein the recombinant host strain is grown in an actively growing culture.

47. A kit comprising the system of any one of claims 1-23.

48. A psilocybin or an intermediate or a side product thereof compound prepared by employing the system of any one of claims 1-23.

49. A psilocybin or an intermediate or a side product thereof compound prepared by the method of any one of claims 24-46.

50. A psilocybin or an intermediate or a side product thereof composition comprising a psilocybin or an intermediate or a side product thereof compound, respectively, prepared by employing the system of any one of claims 1-23, and a carrier and/or an excipient.

51. A psilocybin or an intermediate or a side product thereof composition comprising a psilocybin or an intermediate or a side product thereof compound, respectively, prepared by the method of any one of claims 24-46, and a carrier and/or an excipient.