WO2023015279A1 - Methods for the production of methylated tryptamine derivatives, intermediates or side products - Google Patents

Methods for the production of methylated tryptamine derivatives, intermediates or side products Download PDF

Info

Publication number
WO2023015279A1
WO2023015279A1 PCT/US2022/074579 US2022074579W WO2023015279A1 WO 2023015279 A1 WO2023015279 A1 WO 2023015279A1 US 2022074579 W US2022074579 W US 2022074579W WO 2023015279 A1 WO2023015279 A1 WO 2023015279A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutant
group
promoter
tryptamine
methylated
Prior art date
Application number
PCT/US2022/074579
Other languages
French (fr)
Inventor
John Andrew Jones
Lucas FRIEDBERG
Original Assignee
Miami University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Miami University filed Critical Miami University
Publication of WO2023015279A1 publication Critical patent/WO2023015279A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01049—Amine N-methyltransferase (2.1.1.49)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/11—Aminopeptidases (3.4.11)
    • C12Y304/11001—Leucyl aminopeptidase (3.4.11.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)

Definitions

  • the general inventive concepts relate to the field of medical therapeutics and more particularly to methods for the production of methylated tryptamine derivatives, intermediates or side products.
  • A/TV-Dimethyltryptamine is a tryptophan-derived alkaloid that is naturally present in many plants and animals.
  • DMT binds to serotonin receptors in the brain inducing mind altering changes as a result.
  • DMT has a history of being consumed by several indigenous groups from the Northwestern Amazon for therapeutic purposes. The most recent estimate of the first known use of DMT by humans dates to pre-Colombian times, or about 1 ,000 years ago, based on carbon dating of a leather bag containing a “ritual bundle,” which contained paraphernalia for consuming psychotropic plants (Miller et al., 2019).
  • DMT-containing mixture called ayahuasca
  • ayahuasca which is made from the leaves of the shrub Psychotria viridis, providing the source of DMT, and the vine Banisteriopsis caapi, providing the monoamine oxidase inhibitors (MAOIs) required for DMT to be orally active.
  • MAOIs monoamine oxidase inhibitors
  • DMT derivative 5-methoxy-/V,7V-dirnethyltryptarnine (5-MeO-DMT) and its active metabolite 5-hydroxy-7V,7V-dimethyltryptamine (5-HO-DMT; “bufotenine”) have been traced back thousands of years for their use in ceremonies in Venezuela, Columbia, and Brazil in the form of crushed seeds known as ‘Yopo’ (Artal and Carbera 2007).
  • 5-MeO-DMT also makes up the active ingredient of the venom and parotid gland secretions of Colorado River Toad, Incilius alvarius (Shen et al. 2011).
  • 5-MeO-DMT exhibits hallucinogenic properties upon parenteral administration and conversion to its active metabolite bufotenine.
  • a method for the production of a methylated tryptamine or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, INMT, and combinations thereof; and culturing the host cell.
  • the prokaryotic host cell is contacted with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, PaNMT, INMT, and combinations thereof.
  • the methylated tryptamine is a methylated tryptamine of Formula I: wherein:
  • R 1 is selected from the group consisting of NH2, NHCH3, N(CH3)2, N(CH3)3 + ;
  • R 2 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH 2 , COOH, CHO, CN, SO3, SO4, and PO 4 ;
  • R 3 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH 2 , COOH, CHO, CN, SO3, SO 4 , and PO 4 ;
  • R 4 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH 2 , COOH, CHO, CN, SO3, SO 4 , and PO 4 ;
  • R 5 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH 2 , COOH, CHO, CN, SO3, SO4, and PO 4 ;
  • R 6 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH 2 , COOH, CHO, CN, SO 3 , SO 4 , and PO 4 ;
  • R 7 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO 2 , NH 2 , COOH, CHO, CN, SO 3 , SO 4 , and PO 4 ; and
  • R 8 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO 2 , NH 2 , CHO, COOH, CN, SO3, SO 4 , and PO 4 .
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the PaNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the INMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • 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.
  • the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius.
  • the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, INMT, and combinations thereof, all under control of a single promoter in operon configuration.
  • the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, INMT, and combinations thereof, all under control of a single promoter in operon configuration.
  • 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.
  • the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • 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.
  • the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
  • the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
  • 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.
  • the intermediate of a methylated tryptamine is an indole or derivatized indole, tryptophan or derivatized tryptophan, tryptamine or derivatized tryptamine.
  • the host cell is cultured with a supplement independently selected from the group consisting of indole, serine, threonine, methionine and combinations thereof.
  • the host cell is cultured with a supplement produced by contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, INMT, and combinations thereof; and culturing the host cell in the presence of an indole of Table 2 or Table 3.
  • the supplement is fed continuously to the host cell.
  • the host cell is grown in an actively growing culture.
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof.
  • the prokaryotic host cell is comprises one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, INMT, and combinations thereof.
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the aNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the INMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the recombinant 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.
  • the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius.
  • the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein the one or more methylated tryptamine production genes are under control of a single promoter in operon configuration.
  • the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein the one or more methylated tryptamine production genes are under control of a single promoter in operon configuration.
  • 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.
  • the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • 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.
  • the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
  • the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
  • 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.
  • an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, all under control of a single promoter in operon configuration.
  • the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, all under control of a single promoter in operon configuration.
  • 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.
  • transfection kit comprising the expression vector of any one of the embodiments described herein.
  • an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, FaNMT, IMNT, and combinations thereof, , wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • 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.
  • the expression vector comprises a psiD gene, a psiK gene, a psiM gene, a PaNMT gene and an IMNT gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • the vector comprises a psiD gene, a T zNMT gene and an IMNT gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • 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.
  • an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
  • the vector comprises a psiD gene, a PaNMT gene and an IMNT gene, wherein each gene is under control of a separate promoter in monocistronic configuration.
  • each promoter is independently selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • transfection kit comprising the expression vector of any one of the embodiments described herein.
  • FIG. 1 shows a table providing chemical structures and names based on R group constituents of tryptamine backbone.
  • FIG. 2 shows a metabolic pathway for the production of TV-methylated tryptamines from various starting substrates: glucose, serine and indole, tryptophan, or tryptamine.
  • psiD psilocybin decarboxylase
  • INMT indolethylamine-TV-methyltransferase (INMT)
  • aNMT phenylalkylamine-A-methyltransferase ( aNMT)
  • SAM S-Adenosyl methionine (cosubstrate);
  • SAH S-Adenosyl homocysteine.
  • NMT TV-methyltryptamine
  • DMT N,N- dimethyltryptamine
  • TMT ATV,TV-trimethyltryptamine.
  • FIGs. 3A-3C show a visual representation of the different pH control schemes utilized.
  • FIG. 3 A Starting pH was adjusted at the start of the experiment.
  • FIG. 3B pH was adjusted to 7.5 at the beginning of the experiment and readjusted to 7.5 every 2 h.
  • FIG. 3C pH was maintained at 7.5 for the entirety of the experiment. The portion between the two horizontal lines highlights the observed optimal pH range for methylated tryptamine production.
  • FIG. 4A NMT concentration (mg/L) produced by INMT strain as a function of temperature and pH.
  • FIG. 4B DMT concentration (mg/L) produced by INMT strain as a function of temperature and pH.
  • FIG. 4C NMT concentration (mg/L) produced by aNMT strain as a function of temperature and pH. *Concentrations were attained from samples that were diluted to fit within the linear portion of the MS DMT standard curve. DMT production from PaNMT was not observed under the conditions of this well plate experiment (defined initial pH).
  • FIGs. 5A-5B show NMT and DMT concentration based on promoter strength of key methyltransferase-encoding gene pETM6-SDM2x-INMT (FIG. 5A) pETM6-SDM2x- PaNMT (FIG. 5B).
  • (-) indicates non-pH controlled growth conditions while all other strains were pH-controlled using the medium throughput well plate assay. Concentrations were attained from samples that were diluted to fit within the linear portion of the MS DMT standard curve.
  • FIG. 7 shows an ultraviolet chromatograph comparing the absorbances of a DMT standard (purchased from Cerilliant) and a bioreactor sample.
  • Blue DMT standard;
  • Black bioreactor sample.
  • FIG. 8 shows extracted Ion Channel Mass-spectroscopy peaks. Time is given on the x axis, and total counts are given on the y axis. The first three chromatographs (top down) are taken from bioreactor samples and the final chromatograph is that of a DMT standard (purchased from Cerilliant): NMT (top), DMT (top-middle), TMT (bottom-middle), DMT standard (bottom). Retention times provided in parentheses.
  • FIG. 9 shows NMT, DMT and TMT concentration based on gene configuration and promoter strength of INMT and PsiD.
  • FIG. 9A shows Operon configuration: pETM6- SDM2X-INMT-PsiD and pETM6-PsiD-INMT.
  • FIG. 9B shows Monocistronic configuration: pETM6-SDM2X-INMT-PsiD.
  • FIG. 9C shows Pseudo-operon configuration: pETM6- SDM2x-PsiD-INMT.
  • FIG. 9D shows Pseudo-operon configuration: pETM6-SDM2x-INMT- PsiD.
  • FIG. 11 shows De novo production of NMT, DMT, and TMT by select lead strains.
  • (- ) symbolizes the negative control empty vector, pETM6-SDM2X.
  • T7-I-D T7-INMT-PsiD.
  • FIG. 12 shows 2L Fed-batch bioreactor studies with select strains. Ml 11 was supplemented with 1 g/L tryptophan, while T7-INMT was supplemented with 150 mg/L tryptamine.
  • FIG. 13 shows 5-MeO-NMT and 5-MeO-DMT production by select strains.
  • (-) pETM6-SDM2X.
  • T7-I-D T7-INMT-PsiD. No 5-MeO-TMT was observed.
  • FIG. 14 shows 5-HO-NMT, 5-HO-DMT, and 5-HO-TMT production.
  • (-) pETM6- SDM2X.
  • T7-I-D T7-INMT-PsiD.
  • 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.
  • prokaryotic host cell encompasses any progeny that is not identical due to mutations that occur during replication.
  • 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.
  • the genetic modification comprises integrating the polynucleotide in the genome of the host cell.
  • the polynucleotide is exogenous in the host cell.
  • methylated tryptamine means a tryptamine containing one or more N-methylations.
  • the term “intermediate” of a methylated tryptamine means an intermediate in the production or biosynthesis of a methylated tryptamine, e.g., indole or derivatized indole, tryptophan or derivatized tryptophan, tryptamine or derivatized tryptamine. See, for example, the derivatized indoles of Table 2.
  • a range is intended to comprise every integer or fraction or value within the range.
  • the general inventive concepts are based, in part, on the surprising synergy between increased production through genetic and fermentation means to quickly identify key process parameters required to enable successful scale-up studies culminating in production of a high- value chemical product.
  • a method for the production of a methylated tryptamine or an intermediate or a side product thereof comprises contacting a host cell with at least one methylated tryptamine production gene selected from: psiD, psiK, psiM, PaNMT, INMT, and combinations thereof to form a recombinant cell; culturing the recombinant cell; and obtaining the methylated tryptamine.
  • the at least one methylated tryptamine production gene is selected from: psiD, PaNMT, INMT and combinations thereof.
  • the host cell is a prokaryotic cell.
  • the host cell is an E. coli cell.
  • a method for the production of a methylated tryptamine or an intermediate or a side product thereof comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, INMT, and combinations thereof; and culturing the host cell.
  • the methylated tryptamine production gene is selected from the group consisting of psiD, PaNMT, INMT, and combinations thereof.
  • 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.
  • the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius.
  • the methylated tryptamine is:
  • R 1 is selected from the group consisting of NH2, NHCH3, N(CH3)2, NH(CH 3 ) 2 + , N(CH3)3 + .
  • R1 is selected from the group consisting of NH 2 , NHCH3, N(CH 3 ) 2 , N(CH 3 ) 3 + .
  • R 2 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4.
  • R 2 is selected from the group consisting of H and C1-C5 alkyl.
  • halogen is selected from the group consisting of F, Cl, Br, and I.
  • R 3 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R 3 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I. [0087] Where R 4 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R 4 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I.
  • R 5 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4.
  • R 5 is selected from the group consisting of H, OH, C1-C5 alkoxy and C1-C5 alkyl.
  • R 5 is O(CH3).
  • halogen is selected from the group consisting of F, Cl, Br, and I.
  • R 6 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4.
  • R 6 is selected from the group consisting of H and C1-C5 alkyl.
  • halogen is selected from the group consisting of F, Cl, Br, and I.
  • R 7 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4.
  • R 7 is selected from the group consisting of H and C1-C5 alkyl.
  • halogen is selected from the group consisting of F, Cl, Br, and I.
  • R 8 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, CHO, COOH, CN, SO3, SO4, and PO4.
  • R 8 is selected from the group consisting of H and C1-C5 alkyl.
  • halogen is selected from the group consisting of F, Cl, Br, and I.
  • the methylated tryptamine is N-methyltryptamine, N,N- dimethyltryptamine (DMT), or A/ /N-trimethyltryptamine (TMT).
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 4, 15, 23, 33, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number PPQ70874, KY984102, KY984099, PPQ98758, 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.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 17, 25, 29, 35, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number PPQ70884, KAF8878011.1, KY984103, KY984100, PPQ80976, 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.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 17, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the PaNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the PaNMT comprises the amino acid sequence of Genbank accession number AWJ64115.1 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.
  • the aNMT is encoded by a nucleotide sequence comprising 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.
  • the INMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the INMT comprises the amino acid sequence of Genbank accession number NP 006765 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.
  • the INMT is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene, a psiM gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a aNMT gene, and an INMT gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator.
  • 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.
  • any intermediate of a methylated tryptamine may be produced by any of the methods described herein.
  • the intermediate of a methylated tryptamine is indole or derivatized indole, tryptophan or derivatized tryptophan, tryptamine or derivatized tryptamine.
  • the host cell is cultured with a supplement independently selected from the group consisting of indole, serine, methionine, tryptophan, tryptamine, 5- methoxy indole, 5 -hydroxy indole, and combinations thereof.
  • the supplement is fed continuously to the host cell.
  • the host cell is grown in an actively growing culture. Continuous feeding is accomplished by using a series of syringe and/or peristaltic pumps whose outlet flow is directly connected to the bioreactor. The set point of these supplement addition pumps is adjusted in response to real-time measurement of cell biomass and specific metabolic levels using UV-vis absorption and HPLC analysis, respectively.
  • the fed-batch fermentation process is focused on maximizing production of target metabolites through harnessing the ability of an actively growing and replicating cell culture to regenerate key co-factors and precursors which are critical to the biosynthesis of target metabolites.
  • This process notably does not involve the centrifugal concentration and reconstitution of cell biomass to artificially higher cell density and/or into production media that was not used to build the initial biomass.
  • the production process involves the inoculation of the reactor from an overnight preculture at low optical density, followed by exponential phase growth entering into a fed-batch phase of production, culminating in a high cell density culture.
  • the methylated tryptamine and intermediate or side products are found extracellularly in the fermentation broth.
  • the methylated tryptamine 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.
  • the products can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions methylated tryptamine or any of the intermediate or side products into the organic phase.
  • contaminants from the fermentation broth can be removed through extraction leaving the methylated tryptamine and/or intermediate or side products in the aqueous phase for collection after drying or crystallization procedures.
  • the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 150 mg/L. In some embodiments, the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 120 mg/L. In yet further embodiments, the methods described herein result in a titer of methylated tryptamine of about 1.0 to about 110 mg/L. In certain embodiments, the methods described herein result in a titer of methylated tryptamine of about 2.0 to about 100 mg/L.
  • the methods described herein result in a titer of methylated tryptamine of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mg/L.
  • the methylated tryptamine is NMT and the titer is about 100 mg/L.
  • the methylated tryptamine is DMT and the titer is about 80 mg/L.
  • the methylated tryptamine is TMT and the titer is about 20 mg/L.
  • the methods described herein result in a molar yield of methylated tryptamine of about 1% to about 50%. In some embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 1 % to about 40%. In yet further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 1% to about 30%. In certain embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 10% to about 30%. In further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 20%.
  • the methods described herein result in de novo production of a methylated tryptamine. In certain embodiments, the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 50 mg/L. In some embodiments, the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 20 mg/L. In yet further embodiments, the methods described herein result in a titer of methylated tryptamine of about 1.0 to about 20 mg/L. In certain embodiments, the methods described herein result in a titer of methylated tryptamine of about 2.0 to about 18 mg/L.
  • the methods described herein result in a titer of methylated tryptamine of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18 mg/L.
  • the methylated tryptamine is NMT and the titer is about 11 mg/L.
  • the methylated tryptamine is DMT and the titer is about 14 mg/L.
  • the methylated tryptamine is TMT and the titer is about 6 mg/L.
  • the methods described herein result in a molar yield of methylated tryptamine of about 0.01% to about 20%. In some embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.01% to about 5%. In yet further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.01% to about 1%. In certain embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.05% to about 0.5%. In further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.1%.
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT and INMT and combinations thereof.
  • the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT and INMT and combinations thereof.
  • the recombinant 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.
  • the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius.
  • the methylated tryptamine is a compound of Formula I as described above.
  • the methylated tryptamine is /V-methyltryptamine, /V,7V-dimethyltryptamine (DMT), or 7V,7V,7V-trimethyltryptamine (TMT).
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 4, 15, 23, 33, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number PPQ70874, KY984102, KY984099, PPQ98758, 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.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 17, 25, 29, 35, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number PPQ70884, KAF8878011.1, KY984103, KY984100, PPQ80976, 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.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 17, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the aNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the PaNMT comprises the amino acid sequence of Genbank accession number AWJ64115.1 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.
  • the PaNMT is encoded by a nucleotide sequence comprising 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.
  • the INMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the INMT comprises the amino acid sequence of Genbank accession number NP 006765 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.
  • the INMT is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene, a psiM gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a PaNMT gene and an INMT gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • 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. (See, WO2021/086513, international application no. PCT/US20/051543, which is hereby incorporated by reference in its entirety and for all purposes).
  • 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.
  • a vector for introducing at least one gene associated with production of a methylated tryptamine the gene may be selected from: psiD,psiK, psiM, PaNMT, and INMT and combinations thereof. In some embodiments, the gene is selected from psiD, PaNMT, and INMT and combinations thereof.
  • the methylated tryptamine is a compound of Formula I as described above.
  • the methylated tryptamine is A-methyltryptamine, /V,7V-dimethyltryptamine (DMT), or N,N, TV-trim ethyltryptamine (TMT).
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 4, 15, 23, 33, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number PPQ70874, KY984102, KY984099, PPQ98758, 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.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 17, 25, 29, 35, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number PPQ70884, KAF8878011.1, KY984103, KY984100, PPQ80976, 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.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 17, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the PaNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the PaNMT comprises the amino acid sequence of Genbank accession number AWJ64115.1 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.
  • the PaNMT is encoded by a nucleotide sequence comprising 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.
  • the INMT gene encodes a polypeptide comprising the amino acid sequence of 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.
  • the INMT comprises the amino acid sequence of Genbank accession number NP 006765 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.
  • the INMT is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the expression vector comprises a psiD gene, a psiK gene, a psiM gene, a PaNMT and an INMT gene all under control of a single promoter in operon configuration.
  • the expression vector comprises a psiD gene, a PaNMT and an INMT gene all under control of a single promoter in operon configuration.
  • the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • the expression vector comprises a psiD gene, a PaNMT and an INMT gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • 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. (See, WO2021/086513, international application no. PCT/US20/051543, which is hereby incorporated by reference in its entirety and for all purposes).
  • 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.
  • transfection kit comprising an expression vector as described herein.
  • kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes.
  • container means such as, e.g., vials or test tubes.
  • Each of such container means comprises components or a mixture of components needed to perform a transfection.
  • kits may include, for example, one or more components selected from vectors, cells, reagents, lipid-aggregate forming compounds, transfection enhancers, or biologically active molecules.
  • Example 1 Methylated tryptamine production in E. coli.
  • E. coli DH5a was used to propagate all plasmids, while BL21 starTM (DE3) was used as the host for all chemical production experiments. Plasmid transformations were completed using standard electro and chemical competency protocols as specified. Unless otherwise noted, Andrew’s Magic Media (AMM; He et al., 2015) (without MOPS and tricine), was supplemented with 1 g/L methionine and 150 mg/L of tryptamine or an equimolar concentration (for example 100 mg/L) of different precursor molecules (tryptophan or serine and indole such as 5-hydroxyindole or 5-methoxyindole) depending on the goal of the experiment (FIG. 2).
  • Non-supplemented AMM was used for preculture growth while supplemented AMM was used for production media (He et al., 2015).
  • Luria Broth (LB) was used for plasmid propagation during cloning.
  • the antibiotic ampicillin 80 ug/mL was added to the culture media where appropriate for plasmid selection.
  • the exogenous pathway gene encoding PsiD was taken from a plasmid construct previously reported for psilocybin biosynthesis (Adams et al., 2019).
  • Plasmid construction Human INMT and Ephedra sinica aNMT gene sequences were ordered as linear, double stranded DNA fragments from Genewiz Inc, New Jersey. INMT and PaNMT were codon optimized, PCR amplified, restriction digested with Ndel and Xho , and ligated into a modified ePathBrick expression vector also digested with Ndel and Xhol.
  • IPTG isopropyl P-D-l -thiogalactopyranoside
  • pH-Controlled, Medium Throughput Screening Method Using the standard screening conditions described above, a modified protocol was developed to test chemical production under controlled pH conditions in a medium throughput well plate screening approach. Replicate 48-well plates were set-up in a way to allow for many replicate cultures which could be sacrificed for pH monitoring and empirical pH adjustment purposes. Beginning at time of induction (4 hours post inoculation), 4 mL (2 mLs from 2 sacrificial wells) of the pH control plate were transferred into a falcon tube and the pH was measured using Fisher Scientific AccumetTM AE150 pH benchtop meter. pH of the sacrificial culture is then adjusted by the addition of 10M KOH in 1 uL increments until the desired setpoint was achieved.
  • the total volume of 10M KOH required was recorded and used to inform the adjustment of the pH for the remaining sacrificial controls and experimental cultures in the 48-well plate format using a multichannel pipette.
  • the pH measuring and adjustment procedure described above was performed every two hours over the course of 12 hours post induction unless otherwise noted.
  • Promoter Library Validation Upon constructing and testing promoter library strains, stock cultures were made by combining cultures grown overnight with 30% sterile glycerol to produce a 15% glycerol stock culture in 96 well plates. Once promoter library constructs were screened using the previously mentioned screening methods, a few top performing strains, based on DMT titer, from each library were selected for further screening. Selected strains (BL21 StarTM (DE3)) were streaked from previously described freezer stocks onto an agar plate containing ampicillin. The following day, streaked plates were used to inoculate a 48 well plate for screening outlined in 2.1.5. Following well inoculation, an agar plate containing ampicillin was streaked to preserve well plate cultures.
  • Plasmid DNA of overnight cultures was isolated and purified using Omega Bio-tek E.Z.N.A® Plasmid DNA mini Kit I followed by digestion and gel electrophoresis to confirm expected plasmid construct. Purified plasmid DNA was then transformed into DH5a and plated on agar plates containing ampicillin. Colonies of transformants were used to grow overnight cultures in LB. DH5a overnight cultures were subject to plasmid DNA purification, followed by digestion and gel electrophoresis to confirm expected plasmid construct. Plasmid DNA was transformed back into (BL21 StarTM (DE3) and plated on agar plates containing ampicillin.
  • the entire 50 mL sacrificial culture was pH adjusted with 10 M KOH.
  • the pH probe was rinsed with 70% EtOH between measurements to reduce chance of contamination.
  • Cultures were sampled 24 h post inoculation. Final OD measurements were taken at time of sample collection and compared to determine potential effects of the overlay on cell viability.
  • Cultures were collected in 50mL Falcon tubes and subject to centrifugation at 4696 ref for 10 minutes to separate media and overlay from cells. Following initial separation, media and overlay solutions were separated into multiple ImL tubes and centrifuged at 21000 ref for 10 minutes to separate media and overlay. Media and overlay where analyzed for tryptamine content as described elsewhere herein.
  • pH Stat Bioreactor Screening Once optimal conditions were determined using standard and pH-controlled screening conditions at both 37 °C and 42 °C, DMT production was scaled up using an Eppendorf BioFlol20 bioreactor with a 1.5 L working volume. The cylindrical vessel was mixed by a direct drive shaft containing two Rushton-type impellers positioned equidistance under the liquid surface.
  • the overnight culture of BL21 StarTM (DE3) containing pETM6-SDM2x-lNMT was grown for 12 hr at 37 °C in 50 mL of AMM supplemented with methionine (1 g/L), tryptamine (150 mg/L), and ampicillin (80 ug/mL) in a 250mL non baffled Erlenmeyer flask.
  • the bioreactor contained the same media composition which was used for the overnight culture and was inoculated at a 2% v/v (30 mL into 1.5 L). Temperature was held at a constant 42 °C with a heat jacket and recirculating cooling water, pH was automatically and continuously controlled at either 6.5, 7, 7.5, or 8, with the addition of 10M KOH.
  • FIG. 7 provides a visual representation of NMT and DMT detection as compared with a DMT standard; however, TMT was unable to be detected as a distinct single peak due to overlapping retention times with DMT. This led to the use of the mass spectroscopy extracted ion chromatographs for all titer analysis.
  • DMT analytical standard was ordered from Cerilliant Corporation. The authentic standard was used to create a standard curve through serial dilutions in spent and filtered cell broth and these samples were analyzed using the methods described below. The standard curve was created to determine both the low and high limits of detection and quantification of about 0.05 mg/L NMT and 0.06 mg/L DMT to 3.09 mg/L NMT and 3.34 mg/L DMT respectively for the MS detector. Several DMT standard curves were run to validate quantification by LCMS using extracted ion chromatograms (FIG.
  • Each standard curve displayed a range of linearity (0.05 mg/L NMT and 0.06 mg/L DMT to 3.09 mg/L NMT and 3.34 mg/L DMT) before demonstrating detector saturation.
  • the slope from the linear portion of the DMT standard curves was used to quantify NMT, DMT, and TMT products on a molar basis due to the lack of commercially available standards for NMT and TMT.
  • EIC Mass spectroscopy extracted ion channels
  • the ISQTM EC mass spectrometer equipped with a heated electrospray ionization (HESI) source, was operated in positive mode.
  • the mass spectrometer was supplied > 99% purity nitrogen using Peak Scientific Genius XE 35 laboratory nitrogen generator.
  • the source and detector conditions were as follows: sheath gas pressure of 80.0 psig, auxiliary gas pressure of 9.7 psig, sweep gas pressure of 0.5 psig, foreline vacuum pump pressure of 1.55 Torr, vaporizer temperature of 500 °C, ion transfer tube temperature of 300 °C, source voltage of 3049 V, source current of 15.90 pA.
  • 3A provides a qualitative visual for the time variant pH levels throughout this assay.
  • NMT titer reached a high of 1.16 +/- 0.003 mg/L when grown at 30 °C and having the media pH initially adjusted to 8. DMT production was not observed in FaNMT-expressing E. coli under these conditions.
  • the 150 mg/L tryptamine supplementation was not exhausted during these studies.
  • FIGs. 5A and 5B show the NMT and DMT concentrations observed in the INMT and aNMT promoter library, respectively.
  • use of the T7 promoter yielded the highest concentrations of both NMT and DMT.
  • NMT and DMT production from INMT under the control of the T7 consensus promoter reached titers of 1.79 +/- 0.37 mg/L and 0.14 +/- 0.03 mg/L, respectively (FIG. 5A).
  • FIG. 6 shows the concentrations of NMT, DMT, and TMT as a function of pH. All concentrations represented in FIG. 6 are from bioreactor samples taken 24 h after inoculation.
  • NMT titers from pH 7.5 fermentations reached 11.44 +/- 2.01 mg/L, a 1.5-fold increase over highest NMT titers observed from 48 well plate assays.
  • DMT titers from pH 7.5 fermentations reached 12.73 +/- 3.28 mg/L, which mark a 7.8-fold increase compared to DMT produced from the top performing 48 well plate assays.
  • TMT titers were also observed in the largest quantities from the pH 7.5 fermentations at a value of 6.76 +/- 2.52 mg/L representing the first TMT production observed from a bacterial culture.
  • FIGs. 9A-9D illustrate the success of the promoter library screening in identifying a pathway construct capable of producing more DMT from tryptophan as compared to the T7-INMT-PsiD expressing strain.
  • FIG. 9A shows the combined results of two separate promoter library screens with varied operon gene orientation, specifically, xx5-PsiD-INMT and xx5-INMT-PsiD.
  • the number of strains screened is ranged between 5-10 times the library size.
  • a total of 96 strains were selected and are represented in Figure 9A, with 48 strains selected per operon gene orientation.
  • FIG. 9B represents the monocistronic library screen of 144 strains for the gene orientation INMT-PsiD, and it should be noted that a monocistronic promoter library with the gene construct PsiD-INMT could not be created due to limitations in plasmid construction methods.
  • FIG. 10 shows the comparison in DMT production between top to middle performing strains selected from each promoter library screening presented in FIGs. 9A-9D.
  • T7 -INMT-PsiD was again used as a baseline comparison for the success of the promoter library in increasing DMT titers. All strains, with the exception of M29, 020, and Ml 32 produced significantly more DMT than T7-INMT- PsiD (p ⁇ 0.05).
  • top strains were also tested for their ability to catalyze de novo biosynthesis (FIG. 11). Screening conditions were conserved from previous studies; however, tryptophan was not supplemented into the media, such that glucose represents the sole carbon source for growth and product formation. All selected promoter library strains produced significantly more DMT (p ⁇ 0.05) than the T7-INMT-PsiD strain, with the best strain producing 14 +/- 0.37 mg/L DMT and 31.3 +/- 0.84 mg/L of total methylated tryptamines (FIG. 11).
  • TMT TMT identified in the bioreactor studies presented above has not been well described in terms of its potential psychoactive effects, and has only minimal mention in the peer reviewed literature (Servillo et al. 2012). Due to the structural similarity to the natural product, aeruginascin, it is expected that TMT may have significant psychoactive activity motivating further study to enhance its production and exploration of its pharmacological potential in animal studies. Furthermore, since the biosynthesis of NMT, DMT, and most notably, TMT, was observed to be catalyzed by the human INMT, this indicates that these mono- and trimethylated derivatives may play a currently unstudied role in human health. The development of an E. coZz-based process to facilitate the efficient biosynthesis of these compounds can lead to more focused studies to determine the roles and mechanism of actions for tryptamines in human neurobiology.
  • Benchtop bioreactor fermentation was carried out similarly to previously described bioreactor methods but with the addition of a glucose feed, which previously went unused as initial studies utilized a pH-controlled batch operation paradigm. Glucose concentrations within the fermentation media was monitored using methods outlined above. With the addition of glucose fed batch strategy, we aimed to revisit the viability of DMT production scale-up; as a result, we chose to ferment our T7-INMT strain with tryptamine supplementation at both 37 °C and 42 °C to compare directly to previous bioreactor data (FIG. 6).
  • FIG. 12 shows the end point NMT, DMT and TMT titers observed under a glucose fed batch condition.
  • Strain Mi l l was grown in AMM supplemented with 1 g/L of tryptophan.
  • Strain T7-INMT was grown in AMM supplemented with 150 mg/L tryptamine.
  • strain Pl 17 which contains a pseudo-operon gene construct with the weak G6 promoter controlling the expression of PsiD and the strong C4 promoter controlling INMT expression (G6-PsiD-C4-INMT), as the most effective strain in producing both 5- MeO-DMT and bufotenine from their respective indole precursors (FIG. 2).
  • DMT derivatives we believed that we could further utilize the observed substrate promiscuity in attempt to produce psilocin (4-HO-DMT), the active form of the native mushroom psychedelic psilocybin, by feeding the substrate 4-HO-indole using the same pathway previously described.
  • FIG. 13 shows the production of 5-MeO-NMT and 5-MeO-DMT by select strains with maximum observed titers of 0.67 +/- 0.02 mg/L and 0.23 +/- 0.02 mg/L, respectively, by strain Pl 17.
  • FIG. 14 shows the production of 5-HO-methylatedtryptamines by select strains with a maximum observed titer of 2.64 +/- 0.003 mg/L, 3.58 +/- 0.02 mg/L, and 0.39 +/- 0.05 mg/L of 5-HO-NMT, Bufotenine, and 5-HO-TMT, respectively, also by strain Pl 17.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

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

Description

METHODS FOR THE PRODUCTION OF METHYLATED TRYPTAMINE DERIVATIVES, INTERMEDIATES OR SIDE PRODUCTS
FIELD
[0001] The general inventive concepts relate to the field of medical therapeutics and more particularly to methods for the production of methylated tryptamine derivatives, 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. 63/203,970, filed August 5, 2021, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0003] The contents of the electronic sequence listing (315691-00041.xml; Size: 54,446 bytes; and Date of Creation: August 4, 2022) is herein incorporated by reference in its entirety.
BACKGROUND
[0004] A/TV-Dimethyltryptamine (DMT) is a tryptophan-derived alkaloid that is naturally present in many plants and animals. As a structural analog of serotonin, DMT binds to serotonin receptors in the brain inducing mind altering changes as a result. DMT has a history of being consumed by several indigenous groups from the Northwestern Amazon for therapeutic purposes. The most recent estimate of the first known use of DMT by humans dates to pre-Colombian times, or about 1 ,000 years ago, based on carbon dating of a leather bag containing a “ritual bundle,” which contained paraphernalia for consuming psychotropic plants (Miller et al., 2019). Indigenous groups orally ingested a DMT-containing mixture called ayahuasca, which is made from the leaves of the shrub Psychotria viridis, providing the source of DMT, and the vine Banisteriopsis caapi, providing the monoamine oxidase inhibitors (MAOIs) required for DMT to be orally active. In the west, DMT was first seen in 1931 when Canadian scientist Richard Manske developed a chemical synthesis route starting from a trimethylated indole derivative (Manske, 1931). It wasn’t until 1946 that Oswaldo Conclaves de Lima discovered its natural occurrence in plants (Lima, 1946). The hallucinogenic properties of DMT were not discovered until 1956 when the Hungarian chemist and psychiatrist Stephen Szara extracted DMT from Mimosa hostilis and self-dosed intramuscularly (Szara, 1956). The discoveries made by the aforementioned scientists led to a link between modern science and the historical use of DMT in religious and spiritual practices rooted in the mind-altering effects of the chemical.
[0005] Similarly, DMT derivative 5-methoxy-/V,7V-dirnethyltryptarnine (5-MeO-DMT) and its active metabolite 5-hydroxy-7V,7V-dimethyltryptamine (5-HO-DMT; “bufotenine”), have been traced back thousands of years for their use in ceremonies in Venezuela, Columbia, and Brazil in the form of crushed seeds known as ‘Yopo’ (Artal and Carbera 2007). Additionally, 5-MeO-DMT also makes up the active ingredient of the venom and parotid gland secretions of Colorado River Toad, Incilius alvarius (Shen et al. 2011). Also, like DMT, 5-MeO-DMT exhibits hallucinogenic properties upon parenteral administration and conversion to its active metabolite bufotenine.
[0006] Clinical depression is a highly prevalent and debilitating disorder estimated to effect 4.4% of the global population (“WHO | Depression and Other Common Mental Disorders,” 2017). Presently available treatments for depression have proven to have low response rates, slow onset of efficacy, as well as adverse effects such as increased suicidality, especially in adolescents (Pacher & Kecskemeti, 2005). Altogether, current treatments fail to provide adequate treatment for roughly 60% of patients struggling with this disease (de Osorio et al., 2015; Fava, 2003; Knoth et al., 2010; Penn & Tracy, 2012). Recently, psychoactives, such as DMT, MDMA, ketamine, and psilocybin, have gained interest as potential alternatives to traditional Selective Serotonin Reuptake Inhibitor (SSRI)-based antidepressants such as fluoxetine (Prozac®). Longitudinal studies of effects of ayahuasca consumption on the psyche of ritual users suggest that DMT is not detrimental to psychological well-being and is conversely associated with reduced incidence of mental health issues (Davis et al., 2019;
Anderson et al., 2012; Barbosa et al., 2016; Bouso et al., 2012; Guimaraes dos Santos, 2013). In an open-label clinical trial held at Universidade de Sao Paulo, Ribeirao Preto, Brazil, six patients were administered ayahuasca and showed significant reductions in their depressive scores of up to 82% based on the Hamilton Rating Scale for Depression (HAM-D) and the Montgomery-Asberg Depression Rating Scale (MADRS) (de Osorio et al., 2015). With growing evidence to support DMT’s use as an effective anti-depressant, there is a growing need to develop sustainable, reproducible, and scalable synthesis methods to provide a safe source for pharmaceutical-grade product. Research on psilocybin, another psychedelic structurally similar to serotonin, has gained attention for its antidepressant properties as well. Recently, psilocybin has successfully been produced in vivo in the mold Aspergillus nidulans, yeast Saccharomyces cerevisiae, and bacteria Escherichia coli (Adams et al., 2019; Hoefgen et al., 2018; Milne et al., 2020). The in vivo production of psilocybin has showed potential as a competitor to traditional chemical synthesis on the basis of scalability, cost, and speed of production (Fricke et al., 2019).
[0007] In 1961, the discovery of the methylation of tryptamine and structurally related compounds by a cytosolic S-adenosyl-L-methionine (SAM)-dependent methyltransferase was described in the rabbit lung (Axelrod, 1961). Further studies observed the production of N- methyltryptamine (NMT) and DMT in human and rat brain incubated with tryptamine, revealing the presence of indolethylamine-A-methyltransferase (INMT) activity (Mandell & Morgan, 1971; Saavedra et al., 1973). In 1999, human INMT was successfully cloned, further elucidating the broad presence of methylated tryptamines in human physiology and metabolism (Thompson et al., 1999). Recently, phenylalkylamine JV-methyltransferase (PaNMT) from Ephedra sinica, has demonstrated a broad range of substrate promiscuity in vitro ranging from its native substrate, norephedrine, to indoleamines (Morris et al., 2018).
[0008] There remains a need for methods for the production of methylated tryptamines and intermediates or side products thereof.
SUMMARY
[0009] Provided is a method for the production of a methylated tryptamine or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, INMT, and combinations thereof; and culturing the host cell. [0010] In some embodiments, the prokaryotic host cell is contacted with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, PaNMT, INMT, and combinations thereof.
[0011] In some embodiments, the methylated tryptamine is a methylated tryptamine of Formula I:
Figure imgf000006_0001
wherein:
R1 is selected from the group consisting of NH2, NHCH3, N(CH3)2, N(CH3)3+;
R2 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R3 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R4 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4; R5 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R6 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R7 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4; and
R8 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, CHO, COOH, CN, SO3, SO4, and PO4.
[0012] In some embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0013] In some embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0014] In some embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0015] In some embodiments, the PaNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
[0016] In some embodiments, the INMT gene encodes a polypeptide comprising the amino acid sequence of 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. [0017] In further embodiments, 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.
[0018] In further embodiments, the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius.
[0019] In some embodiments, the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, INMT, and combinations thereof, all under control of a single promoter in operon configuration. In some embodiments, the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, INMT, and combinations thereof, all under control of a single promoter in operon configuration.
[0020] 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.
[0021] In some embodiments, the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration. In some embodiments, the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
[0022] In further embodiment, 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 prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration. In some embodiments, the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
[0024] 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.
[0025] In some embodiments, the intermediate of a methylated tryptamine is an indole or derivatized indole, tryptophan or derivatized tryptophan, tryptamine or derivatized tryptamine.
[0026] In some embodiments, the host cell is cultured with a supplement independently selected from the group consisting of indole, serine, threonine, methionine and combinations thereof.
[0027] In some embodiments, the host cell is cultured with a supplement produced by contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, INMT, and combinations thereof; and culturing the host cell in the presence of an indole of Table 2 or Table 3. In further embodiments, the supplement is fed continuously to the host cell.
[0028] In some embodiments, the host cell is grown in an actively growing culture.
[0029] Also provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof.
[0030] In some embodiments, the prokaryotic host cell is comprises one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, INMT, and combinations thereof. [0031] In some embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0032] In some embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0033] In some embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0034] In some embodiments, the aNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
[0035] In some embodiments, the INMT gene encodes a polypeptide comprising the amino acid sequence of 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.
[0036] In some embodiments, the recombinant 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.
[0037] In further embodiments, the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius. [0038] In some embodiments, the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein the one or more methylated tryptamine production genes are under control of a single promoter in operon configuration. In some embodiments, the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein the one or more methylated tryptamine production genes are under control of a single promoter in operon configuration.
[0039] 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.
[0040] In some embodiments, the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration. In some embodiments, the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
[0041] 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 expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration. In some embodiments, the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration. [0043] 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.
[0044] Provided is an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, all under control of a single promoter in operon configuration. In some embodiments, the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT, IMNT, and combinations thereof, all under control of a single promoter in operon configuration.
[0045] 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.
[0046] Provided is a transfection kit comprising the expression vector of any one of the embodiments described herein.
[0047] Provided is an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration. In some embodiments, the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, FaNMT, IMNT, and combinations thereof, , wherein each gene is under control of a separate promoter in pseudooperon configuration.
[0048] 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.
[0049] In some embodiments, the expression vector comprises a psiD gene, a psiK gene, a psiM gene, a PaNMT gene and an IMNT gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In some embodiments, the vector comprises a psiD gene, a T zNMT gene and an IMNT gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. [0050] 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.
[0051] Provided is an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration. In some embodiments, the vector comprises a psiD gene, a PaNMT gene and an IMNT gene, wherein each gene is under control of a separate promoter in monocistronic configuration.
[0052] In some embodiments, each promoter is independently selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0053] Provided is a transfection kit comprising the expression vector of any one of the embodiments described herein.
DESCRIPTION OF THE FIGURES
[0054] FIG. 1 shows a table providing chemical structures and names based on R group constituents of tryptamine backbone.
[0055] FIG. 2 shows a metabolic pathway for the production of TV-methylated tryptamines from various starting substrates: glucose, serine and indole, tryptophan, or tryptamine. psiD = psilocybin decarboxylase; INMT = indolethylamine-TV-methyltransferase (INMT); aNMT = phenylalkylamine-A-methyltransferase ( aNMT); SAM = S-Adenosyl methionine (cosubstrate); SAH = S-Adenosyl homocysteine. NMT = TV-methyltryptamine; DMT = N,N- dimethyltryptamine; TMT = ATV,TV-trimethyltryptamine.
[0056] FIGs. 3A-3C show a visual representation of the different pH control schemes utilized. FIG. 3 A: Starting pH was adjusted at the start of the experiment. FIG. 3B: pH was adjusted to 7.5 at the beginning of the experiment and readjusted to 7.5 every 2 h. FIG. 3C: pH was maintained at 7.5 for the entirety of the experiment. The portion between the two horizontal lines highlights the observed optimal pH range for methylated tryptamine production.
[0057] FIGs. 4A-4C show NMT and DMT concentrations from BL21 Star™ (DE3) pETM6- SDM2x-INMT and pETM6-SDM2x-7’aNMT plasmid expressing bacteria from 48 well plates at t = 24 h post inoculation. FIG. 4A: NMT concentration (mg/L) produced by INMT strain as a function of temperature and pH. FIG. 4B: DMT concentration (mg/L) produced by INMT strain as a function of temperature and pH. FIG. 4C: NMT concentration (mg/L) produced by aNMT strain as a function of temperature and pH. *Concentrations were attained from samples that were diluted to fit within the linear portion of the MS DMT standard curve. DMT production from PaNMT was not observed under the conditions of this well plate experiment (defined initial pH).
[0058] FIGs. 5A-5B show NMT and DMT concentration based on promoter strength of key methyltransferase-encoding gene pETM6-SDM2x-INMT (FIG. 5A) pETM6-SDM2x- PaNMT (FIG. 5B). (-) indicates non-pH controlled growth conditions while all other strains were pH-controlled using the medium throughput well plate assay. Concentrations were attained from samples that were diluted to fit within the linear portion of the MS DMT standard curve.
[0059] FIG. 6 shows NMT, DMT and TMT concentrations from pH stat bioreactor runs of BL21 star (DE3) pETM6-SDM2x-INMT. Highest concentrations of NMT, DMT, and TMT were achieved at pH = 7.5. Concentrations were attained from samples that were diluted to fit within the linear portion of the MS DMT standard curve.
[0060] FIG. 7 shows an ultraviolet chromatograph comparing the absorbances of a DMT standard (purchased from Cerilliant) and a bioreactor sample. Blue = DMT standard; Black = bioreactor sample.
[0061] FIG. 8 shows extracted Ion Channel Mass-spectroscopy peaks. Time is given on the x axis, and total counts are given on the y axis. The first three chromatographs (top down) are taken from bioreactor samples and the final chromatograph is that of a DMT standard (purchased from Cerilliant): NMT (top), DMT (top-middle), TMT (bottom-middle), DMT standard (bottom). Retention times provided in parentheses. [0062] FIG. 9 shows NMT, DMT and TMT concentration based on gene configuration and promoter strength of INMT and PsiD. FIG. 9A shows Operon configuration: pETM6- SDM2X-INMT-PsiD and pETM6-PsiD-INMT. FIG. 9B shows Monocistronic configuration: pETM6-SDM2X-INMT-PsiD. FIG. 9C shows Pseudo-operon configuration: pETM6- SDM2x-PsiD-INMT. FIG. 9D shows Pseudo-operon configuration: pETM6-SDM2x-INMT- PsiD.
[0063] FIG. 10 shows NMT and DMT production of top performing strains selected from each distinct library construct. Strain: M = monocistronic, P = pseudo-operon, O = operon.
[0064] FIG. 11 shows De novo production of NMT, DMT, and TMT by select lead strains. (- ) symbolizes the negative control empty vector, pETM6-SDM2X. T7-I-D = T7-INMT-PsiD.
[0065] FIG. 12 shows 2L Fed-batch bioreactor studies with select strains. Ml 11 was supplemented with 1 g/L tryptophan, while T7-INMT was supplemented with 150 mg/L tryptamine.
[0066] FIG. 13 shows 5-MeO-NMT and 5-MeO-DMT production by select strains. (-) = pETM6-SDM2X. T7-I-D = T7-INMT-PsiD. No 5-MeO-TMT was observed.
[0067] FIG. 14 shows 5-HO-NMT, 5-HO-DMT, and 5-HO-TMT production. (-) = pETM6- SDM2X. T7-I-D = T7-INMT-PsiD.
DETAILED DESCRIPTION
[0068] 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.
[0069] 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. [0070] 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.
[0071] “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.
[0072] 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.
[0073] 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.
[0074] As used herein, the term “methylated tryptamine” means a tryptamine containing one or more N-methylations.
[0075] As used herein, the term “intermediate” of a methylated tryptamine means an intermediate in the production or biosynthesis of a methylated tryptamine, e.g., indole or derivatized indole, tryptophan or derivatized tryptophan, tryptamine or derivatized tryptamine. See, for example, the derivatized indoles of Table 2.
[0076] In some embodiments of any of the compositions or methods described herein, a range is intended to comprise every integer or fraction or value within the range.
[0077] The materials, compositions, and methods described herein are intended to be used to provide novel routes for the production of methylated tryptamines and intermediates.
[0078] Despite advances in the chemical synthesis of methylated tryptamines, 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 methylated tryptamines.
[0079] There is an unmet need for large scale production and isolation of methylated tryptamines.
I. Methods, vectors, host cells and kits for the production of a methylated tryptamine or an intermediate or a side product thereof
Methods
[0080] The general inventive concepts are based, in part, on the surprising synergy between increased production through genetic and fermentation means to quickly identify key process parameters required to enable successful scale-up studies culminating in production of a high- value chemical product.
[0081] Provided is a method for the production of a methylated tryptamine or an intermediate or a side product thereof. The method comprises contacting a host cell with at least one methylated tryptamine production gene selected from: psiD, psiK, psiM, PaNMT, INMT, and combinations thereof to form a recombinant cell; culturing the recombinant cell; and obtaining the methylated tryptamine. In some embodiments, the at least one methylated tryptamine production gene is selected from: psiD, PaNMT, INMT and combinations thereof. In certain embodiments, the host cell is a prokaryotic cell. In certain exemplary embodiments, the host cell is an E. coli cell.
[0082] Provided is a method for the production of a methylated tryptamine or an intermediate or a side product thereof comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, INMT, and combinations thereof; and culturing the host cell. In some embodiments, the methylated tryptamine production gene is selected from the group consisting of psiD, PaNMT, INMT, and combinations thereof. In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In further embodiments, the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius.
[0083] In some embodiments, the methylated tryptamine is:
Figure imgf000018_0001
Formula I
[0084] Where R1 is selected from the group consisting of NH2, NHCH3, N(CH3)2, NH(CH3)2 +, N(CH3)3+. In further embodiments, R1 is selected from the group consisting of NH2, NHCH3, N(CH3)2, N(CH3)3 +.
[0085] Where R2 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R2 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I.
[0086] Where R3 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R3 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I. [0087] Where R4 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R4 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I.
[0088] Where R5 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R5 is selected from the group consisting of H, OH, C1-C5 alkoxy and C1-C5 alkyl. In yet further embodiments, R5 is O(CH3). In further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I.
[0089] Where R6 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R6 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I.
[0090] Where R7 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4. In further embodiments, R7 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I.
[0091] Where R8 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, CHO, COOH, CN, SO3, SO4, and PO4. In further embodiments, R8 is selected from the group consisting of H and C1-C5 alkyl. In yet further embodiments, halogen is selected from the group consisting of F, Cl, Br, and I.
[0092] In some embodiments, the methylated tryptamine is N-methyltryptamine, N,N- dimethyltryptamine (DMT), or A/ /N-trimethyltryptamine (TMT).
[0093] In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 4, 15, 23, 33, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0094] In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number PPQ70874, KY984102, KY984099, PPQ98758, 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: 17, 25, 29, 35, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0095] In certain embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number PPQ70884, KAF8878011.1, KY984103, KY984100, PPQ80976, 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: 17, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0096] In certain embodiments, the PaNMT gene encodes a polypeptide comprising the amino acid sequence of 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. In certain embodiments, the PaNMT comprises the amino acid sequence of Genbank accession number AWJ64115.1 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 aNMT is encoded by a nucleotide sequence comprising 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.
[0097] In certain embodiments, the INMT gene encodes a polypeptide comprising the amino acid sequence of 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. In certain embodiments, the INMT comprises the amino acid sequence of Genbank accession number NP 006765 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 INMT is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0098] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene, a psiM gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a aNMT gene, and an INMT 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. (See, WO2021/086513, international application no. PCT/US20/051543, which is hereby incorporated by reference in its entirety and for all purposes). [0099] 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.
[0100] It is envisaged that any intermediate of a methylated tryptamine may be produced by any of the methods described herein. In some embodiments, the intermediate of a methylated tryptamine is indole or derivatized indole, tryptophan or derivatized tryptophan, tryptamine or derivatized tryptamine.
[0101] In certain embodiments, the host cell is cultured with a supplement independently selected from the group consisting of indole, serine, methionine, tryptophan, tryptamine, 5- methoxy indole, 5 -hydroxy indole, and combinations thereof. In certain exemplary embodiments, the supplement is fed continuously to the host cell. In further embodiments, the host cell is grown in an actively growing culture. Continuous feeding is accomplished by using a series of syringe and/or peristaltic pumps whose outlet flow is directly connected to the bioreactor. The set point of these supplement addition pumps is adjusted in response to real-time measurement of cell biomass and specific metabolic levels using UV-vis absorption and HPLC analysis, respectively. The fed-batch fermentation process is focused on maximizing production of target metabolites through harnessing the ability of an actively growing and replicating cell culture to regenerate key co-factors and precursors which are critical to the biosynthesis of target metabolites. This process notably does not involve the centrifugal concentration and reconstitution of cell biomass to artificially higher cell density and/or into production media that was not used to build the initial biomass. The production process involves the inoculation of the reactor from an overnight preculture at low optical density, followed by exponential phase growth entering into a fed-batch phase of production, culminating in a high cell density culture.
[0102] The methylated tryptamine and intermediate or side products are found extracellularly in the fermentation broth. In certain embodiments, the methylated tryptamine 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 methylated tryptamine 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 methylated tryptamine and/or intermediate or side products in the aqueous phase for collection after drying or crystallization procedures.
[0103] In certain embodiments, the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 150 mg/L. In some embodiments, the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 120 mg/L. In yet further embodiments, the methods described herein result in a titer of methylated tryptamine of about 1.0 to about 110 mg/L. In certain embodiments, the methods described herein result in a titer of methylated tryptamine of about 2.0 to about 100 mg/L. In further embodiments, the methods described herein result in a titer of methylated tryptamine of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mg/L. In some embodiments, the methylated tryptamine is NMT and the titer is about 100 mg/L. In some embodiments, the methylated tryptamine is DMT and the titer is about 80 mg/L. In some embodiments, the methylated tryptamine is TMT and the titer is about 20 mg/L.
[0104] In certain embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 1% to about 50%. In some embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 1 % to about 40%. In yet further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 1% to about 30%. In certain embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 10% to about 30%. In further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 20%.
Yields for de novo production of a methylated tryptamine
[0105] In certain embodiments, the methods described herein result in de novo production of a methylated tryptamine. In certain embodiments, the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 50 mg/L. In some embodiments, the methods described herein result in a titer of methylated tryptamine of about 0.5 to about 20 mg/L. In yet further embodiments, the methods described herein result in a titer of methylated tryptamine of about 1.0 to about 20 mg/L. In certain embodiments, the methods described herein result in a titer of methylated tryptamine of about 2.0 to about 18 mg/L. In further embodiments, the methods described herein result in a titer of methylated tryptamine of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18 mg/L. In some embodiments, the methylated tryptamine is NMT and the titer is about 11 mg/L. In some embodiments, the methylated tryptamine is DMT and the titer is about 14 mg/L. In some embodiments, the methylated tryptamine is TMT and the titer is about 6 mg/L.
[0106] In certain embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.01% to about 20%. In some embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.01% to about 5%. In yet further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.01% to about 1%. In certain embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.05% to about 0.5%. In further embodiments, the methods described herein result in a molar yield of methylated tryptamine of about 0.1%.
Recombinant prokaryotic cells for the production of a methylated tryptamine or an intermediate or a side product thereof
[0107] Provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT and INMT and combinations thereof. In some embodiments, the vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, aNMT and INMT and combinations thereof.
[0108] In certain embodiments, the recombinant 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.
[0109] In further embodiments, the methylated tryptamine production gene is from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, Gymnopilus dilepis, or Gymnopilus junonius. [0110] In some embodiments, the methylated tryptamine is a compound of Formula I as described above. In further embodiments, the methylated tryptamine is /V-methyltryptamine, /V,7V-dimethyltryptamine (DMT), or 7V,7V,7V-trimethyltryptamine (TMT).
[0111] In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 4, 15, 23, 33, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0112] In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number PPQ70874, KY984102, KY984099, PPQ98758, 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: 17, 25, 29, 35, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0113] In certain embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number PPQ70884, KAF8878011.1, KY984103, KY984100, PPQ80976, 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: 17, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0114] In certain embodiments, the aNMT gene encodes a polypeptide comprising the amino acid sequence of 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. In certain embodiments, the PaNMT comprises the amino acid sequence of Genbank accession number AWJ64115.1 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 PaNMT is encoded by a nucleotide sequence comprising 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.
[0115] In certain embodiments, the INMT gene encodes a polypeptide comprising the amino acid sequence of 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. In certain embodiments, the INMT comprises the amino acid sequence of Genbank accession number NP 006765 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 INMT is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0116] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene, a psiM gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a PaNMT gene and an INMT gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a PaNMT gene and an INMT 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. (See, WO2021/086513, international application no. PCT/US20/051543, which is hereby incorporated by reference in its entirety and for all purposes).
[0117] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
Expression vectors
[0118] Provided is a vector for introducing at least one gene associated with production of a methylated tryptamine; the gene may be selected from: psiD,psiK, psiM, PaNMT, and INMT and combinations thereof. In some embodiments, the gene is selected from psiD, PaNMT, and INMT and combinations thereof.
[0119] In some embodiments, the methylated tryptamine is a compound of Formula I as described above. In further embodiments, the methylated tryptamine is A-methyltryptamine, /V,7V-dimethyltryptamine (DMT), or N,N, TV-trim ethyltryptamine (TMT).
[0120] In certain embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, PPQ70875, KY984104, PPQ80975, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 4, 15, 23, 33, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. [0121] In certain embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number PPQ70874, KY984102, KY984099, PPQ98758, 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: 17, 25, 29, 35, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0122] In certain embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number PPQ70884, KAF8878011.1, KY984103, KY984100, PPQ80976, 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: 17, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0123] In certain embodiments, the PaNMT gene encodes a polypeptide comprising the amino acid sequence of 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. In certain embodiments, the PaNMT comprises the amino acid sequence of Genbank accession number AWJ64115.1 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 PaNMT is encoded by a nucleotide sequence comprising 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. [0124] In certain embodiments, the INMT gene encodes a polypeptide comprising the amino acid sequence of 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. In certain embodiments, the INMT comprises the amino acid sequence of Genbank accession number NP 006765 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 INMT is encoded by a nucleotide sequence comprising SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0125] In certain embodiments, the expression vector comprises a psiD gene, a psiK gene, a psiM gene, a PaNMT and an INMT gene all under control of a single promoter in operon configuration. In certain embodiments, the expression vector comprises a psiD gene, a PaNMT and an INMT gene all under control of a single promoter in operon configuration. In certain embodiments, the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PaNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, the expression vector comprises a psiD gene, a PaNMT and an INMT 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. (See, WO2021/086513, international application no. PCT/US20/051543, which is hereby incorporated by reference in its entirety and for all purposes).
[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.
Kits
[0127] Provided is a transfection kit comprising an expression vector as described herein.
Such a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes. Each of such container means comprises components or a mixture of components needed to perform a transfection. Such kits may include, for example, one or more components selected from vectors, cells, reagents, lipid-aggregate forming compounds, transfection enhancers, or biologically active molecules.
EXAMPLES
[0128] The following examples describe various compositions and methods for genetic modification of cells to aid in the production of methylated tryptamine, according to the general inventive concepts.
Example 1; Methylated tryptamine production in E. coli.
Materials and Methods
Bacterial strains, vectors, and media
[0129] E. coli DH5a was used to propagate all plasmids, while BL21 star™ (DE3) was used as the host for all chemical production experiments. Plasmid transformations were completed using standard electro and chemical competency protocols as specified. Unless otherwise noted, Andrew’s Magic Media (AMM; He et al., 2015) (without MOPS and tricine), was supplemented with 1 g/L methionine and 150 mg/L of tryptamine or an equimolar concentration (for example 100 mg/L) of different precursor molecules (tryptophan or serine and indole such as 5-hydroxyindole or 5-methoxyindole) depending on the goal of the experiment (FIG. 2). Non-supplemented AMM was used for preculture growth while supplemented AMM was used for production media (He et al., 2015). Luria Broth (LB) was used for plasmid propagation during cloning. The antibiotic ampicillin (80 ug/mL) was added to the culture media where appropriate for plasmid selection. The exogenous pathway gene encoding PsiD was taken from a plasmid construct previously reported for psilocybin biosynthesis (Adams et al., 2019).
[0130] Plasmid construction: Human INMT and Ephedra sinica aNMT gene sequences were ordered as linear, double stranded DNA fragments from Genewiz Inc, New Jersey. INMT and PaNMT were codon optimized, PCR amplified, restriction digested with Ndel and Xho , and ligated into a modified ePathBrick expression vector also digested with Ndel and Xhol. Plasmids containing the genes responsible for INMT and PaNMT expression, #4 and #5 respectively, were digested, gel extracted, and ligated, using the restriction enzymes Bcul and Sall, to a DNA fragment from plasmid #19, which carries the psiD expressing gene (Adams et al, 2019), which was digested using Xbal and Sall. The resulting plasmids, #6 and #7, respectively, contain psiD and either INMT or aNMT in pseudo-operon configuration (Table 1). All multigene expression plasmids were constructed using a modified version of the previously published ePathBrick methods as described above, while all transcriptional libraries were constructed using standard ePathOptimize methods and mutant T7 promoters G6, H9, H10, and C4 (Jones et al., 2015; Xu et al., 2012).
[0131] Standard screening conditions: Standard screening was performed in 2 ml working volume cultures in 48-well plates at 37 °C. AMM with aforementioned supplements was used for both overnight and production cultures. Overnight cultures were grown either from agar plates or freezer stock culture in AMM (pH = 7.0) with appropriate antibiotics for 12-16 h in a shaking (250 rpm) incubator at 37 °C or 42 °C. Production cultures were grown in media identical to media used for overnight cultures and were inoculated with the overnight cultures at a 2% working volume (40 pL) and utilized the same incubator conditions as the overnight cultures. Induction with 1 mM isopropyl P-D-l -thiogalactopyranoside (IPTG) occurred 4h after inoculation unless otherwise noted. Cultures were then sampled 24 h post inoculation. Samples were subjected to HPLC and LC-MS analysis as described below.
[0132] Initial pH screening method'. The standard screening method outlined above was used with the addition of varying the initial pH condition. The overnight cultures were grown using the conditions outlined above to result in a standardized inoculum. Before inoculation of the production cultures, the AMM media, supplemented with 150 mg/L tryptamine, 1 g/L methionine, and ampicillin, was pH adjusted to either 7.0, 7.5, or 8.0 using IM KOH, or to 6.0 or 6.5 using IM HC1. Fermentation pH was not monitored throughout the fermentation and conditions correspond to those outlined in the standard screening method outlined above.
[0133] pH-Controlled, Medium Throughput Screening Method'. Using the standard screening conditions described above, a modified protocol was developed to test chemical production under controlled pH conditions in a medium throughput well plate screening approach. Replicate 48-well plates were set-up in a way to allow for many replicate cultures which could be sacrificed for pH monitoring and empirical pH adjustment purposes. Beginning at time of induction (4 hours post inoculation), 4 mL (2 mLs from 2 sacrificial wells) of the pH control plate were transferred into a falcon tube and the pH was measured using Fisher Scientific Accumet™ AE150 pH benchtop meter. pH of the sacrificial culture is then adjusted by the addition of 10M KOH in 1 uL increments until the desired setpoint was achieved. The total volume of 10M KOH required was recorded and used to inform the adjustment of the pH for the remaining sacrificial controls and experimental cultures in the 48-well plate format using a multichannel pipette. The pH measuring and adjustment procedure described above was performed every two hours over the course of 12 hours post induction unless otherwise noted.
[0134] Promoter Library Validation'. Upon constructing and testing promoter library strains, stock cultures were made by combining cultures grown overnight with 30% sterile glycerol to produce a 15% glycerol stock culture in 96 well plates. Once promoter library constructs were screened using the previously mentioned screening methods, a few top performing strains, based on DMT titer, from each library were selected for further screening. Selected strains (BL21 Star™ (DE3)) were streaked from previously described freezer stocks onto an agar plate containing ampicillin. The following day, streaked plates were used to inoculate a 48 well plate for screening outlined in 2.1.5. Following well inoculation, an agar plate containing ampicillin was streaked to preserve well plate cultures. Once strain performance was validated, select colonies from the 48 well agar plate streak were grown overnight in AMM. Plasmid DNA of overnight cultures was isolated and purified using Omega Bio-tek E.Z.N.A® Plasmid DNA mini Kit I followed by digestion and gel electrophoresis to confirm expected plasmid construct. Purified plasmid DNA was then transformed into DH5a and plated on agar plates containing ampicillin. Colonies of transformants were used to grow overnight cultures in LB. DH5a overnight cultures were subject to plasmid DNA purification, followed by digestion and gel electrophoresis to confirm expected plasmid construct. Plasmid DNA was transformed back into (BL21 Star™ (DE3) and plated on agar plates containing ampicillin. Transformants were grown overnight in AMM media. Overnight cultures were used to create freezer stocks, which served as the final production strain moving forward with additional screening. [0135] Overlay Screening Method and Sample Collection'. A modified version of the pH controlled; medium throughput screening method outlined above was used to test efficacy of hydrocarbon overlays on DMT production. 250mL Erlenmeyer flasks were used to test the DMT production of 50mL cultures with the addition of lOmL of a chemical overlay (Jang et al. 2011). Two chemical overlays, dodecane and diisononyl phthalate (DINP), were tested individually. As mentioned above, additional cultures were grown to be used for sacrificial pH measurements to inform pH adjustment of experimental cultures. To reduce error in pH adjusting cultures, the entire 50 mL sacrificial culture was pH adjusted with 10 M KOH. The pH probe was rinsed with 70% EtOH between measurements to reduce chance of contamination. Cultures were sampled 24 h post inoculation. Final OD measurements were taken at time of sample collection and compared to determine potential effects of the overlay on cell viability. Cultures were collected in 50mL Falcon tubes and subject to centrifugation at 4696 ref for 10 minutes to separate media and overlay from cells. Following initial separation, media and overlay solutions were separated into multiple ImL tubes and centrifuged at 21000 ref for 10 minutes to separate media and overlay. Media and overlay where analyzed for tryptamine content as described elsewhere herein.
[0136] pH Stat Bioreactor Screening'. Once optimal conditions were determined using standard and pH-controlled screening conditions at both 37 °C and 42 °C, DMT production was scaled up using an Eppendorf BioFlol20 bioreactor with a 1.5 L working volume. The cylindrical vessel was mixed by a direct drive shaft containing two Rushton-type impellers positioned equidistance under the liquid surface. The overnight culture of BL21 Star™ (DE3) containing pETM6-SDM2x-lNMT was grown for 12 hr at 37 °C in 50 mL of AMM supplemented with methionine (1 g/L), tryptamine (150 mg/L), and ampicillin (80 ug/mL) in a 250mL non baffled Erlenmeyer flask. The bioreactor contained the same media composition which was used for the overnight culture and was inoculated at a 2% v/v (30 mL into 1.5 L). Temperature was held at a constant 42 °C with a heat jacket and recirculating cooling water, pH was automatically and continuously controlled at either 6.5, 7, 7.5, or 8, with the addition of 10M KOH. Agitation and air flow rate were maintained at 500 rpm and 2 v/v (3 SLPM) for the entirety of the 24-hour fermentation. Samples were collected periodically for measurement of ODeoo and metabolite analysis. The bioreactor was induced with 1 mM 1PTG 4 hours post inoculation. Apart from periodic sample collection, the reactor screening process required minimal observation with integrated control systems sensing and maintaining process parameters as described above. The concentration of DMT and all metabolic intermediates and side products were analyzed via HPLC and LC-MS.
[0137] Standard Curve Development’. Quantification using absorbance in the ultraviolet region (e.g., 280 nm) was not utilized in this study due to difficulty achieving separation of key metabolites using liquid chromatography. FIG. 7 provides a visual representation of NMT and DMT detection as compared with a DMT standard; however, TMT was unable to be detected as a distinct single peak due to overlapping retention times with DMT. This led to the use of the mass spectroscopy extracted ion chromatographs for all titer analysis.
[0138] DMT analytical standard was ordered from Cerilliant Corporation. The authentic standard was used to create a standard curve through serial dilutions in spent and filtered cell broth and these samples were analyzed using the methods described below. The standard curve was created to determine both the low and high limits of detection and quantification of about 0.05 mg/L NMT and 0.06 mg/L DMT to 3.09 mg/L NMT and 3.34 mg/L DMT respectively for the MS detector. Several DMT standard curves were run to validate quantification by LCMS using extracted ion chromatograms (FIG. 8): 1) An undiluted bioreactor broth sample containing DMT was initially spiked with 40 mg/L of pure DMT to determine if DMT saturation affected the accuracy of the peak area of the the Extracted Ion Channels (EIC) for NMT and TMT, 2) An undiluted bioreactor broth sample containing DMT was serially diluted in a negative control cell broth that was free of DMT and each diluted sample was spiked with 40 mg/L of pure DMT to ensure that saturation of DMT would not affect the peak areas of NMT or TMT extracted ion channels of the diluted samples, 3) A traditional standard curve was made by diluting pure DMT in negative control cell broth to ensure that “matrix” effects did not affect peak quantification. Each standard curve displayed a range of linearity (0.05 mg/L NMT and 0.06 mg/L DMT to 3.09 mg/L NMT and 3.34 mg/L DMT) before demonstrating detector saturation. The slope from the linear portion of the DMT standard curves was used to quantify NMT, DMT, and TMT products on a molar basis due to the lack of commercially available standards for NMT and TMT.
[0139] Analytical Methods’. Samples were prepared for HPLC and LC-MS analysis by centrifugation at 21,000 ref for 5 minutes; 2 pL of the resulting supernatant was then injected for analysis. Analysis was performed on a Thermo Scientific Ultimate 3000 High- Performance Liquid Chromatography (HPLC) system equipped with Diode Array Detector (DAD) and Thermo Scientific ISQ™ EC single quadrupole mass spectrometer (MS).
[0140] Mass spectroscopy extracted ion channels (EIC) were used to quantify the aromatic compounds of interest in this study (i.e., tryptamine, NMT, DMT, and TMT). Metabolite separation was performed using an Agilent Zorbax Eclipse XDB-C18 analytical column (3.0 mm x 250 mm, 5 pm) with mobile phases of water (A) and acetonitrile (B) both containing 0.1% formic acid at a rate of 1 mL/min: 0 min, 5% B; 0.43 min, 5% B; 5.15 min, 19% B;
6.44 min, 100% B; 7.73 min, 100% B; 7.73 min, 5% B; 9.87 min, 5% B. The ISQ™ EC mass spectrometer, equipped with a heated electrospray ionization (HESI) source, was operated in positive mode. The mass spectrometer was supplied > 99% purity nitrogen using Peak Scientific Genius XE 35 laboratory nitrogen generator. The source and detector conditions were as follows: sheath gas pressure of 80.0 psig, auxiliary gas pressure of 9.7 psig, sweep gas pressure of 0.5 psig, foreline vacuum pump pressure of 1.55 Torr, vaporizer temperature of 500 °C, ion transfer tube temperature of 300 °C, source voltage of 3049 V, source current of 15.90 pA.
[0141] LC-MS data was collected, where the full MS scan was used to provide an extracted ion chromatogram (EIC) of our compounds of interest (M+l): Tryptamine (m/z 161), NMT (m/z 175), DMT (m/z 189), TMT (m/z 203). This method resulted in the following observed retention times as verified by analytical standards (when commercially available): tryptamine (5.63 min), NMT (5.79 min), DMT (6.01 min) and TMT (6.05 min), FIG. 7. Samples quantified by LC-MS were appropriately diluted such that their detector response falls in the linear response regime, as reported above. All data was managed and processed using Thermo Scientific Chromeleon 7.3 Chromatography Data System.
Results
[0142] Temperature and pH Dependent Production ofN,N-dimethyltryptamine\ Both methyltransferases investigated herewith, INMT and PaNMT, were expressed in E. coli using the strong consensus Tl-lac promoter system. The production of NMT and DMT by these recombinant E. coli hosts were monitored as a function of both temperature and pH. In this assessment, the pathway intermediate, tryptamine, was provided in the culture media to allow for the direct assessment of effective methyltransferase activity. Induction with IPTG led to the observation of the presence of NMT and DMT in both INMT- and PaNMT-expressing E. coli under well-plate conditions. DMT was never observed in the absence of NMT. FIG. 3A provides a qualitative visual for the time variant pH levels throughout this assay. The highest titers for both NMT and DMT, observed at 7.58 +/- 0.51 mg/L and 0.22 +/- 0.01 mg/L respectively, were observed in INMT-expressing E. coli at 42 °C with an initial media pH of 8.0. NMT and DMT concentrations were significantly higher in cultures incubated with a starting pH of 8 compared to other pHs tested (p < 0.05) (FIG. 4). In 7’aNMT-expressing E. coli, NMT titer reached a high of 1.16 +/- 0.003 mg/L when grown at 30 °C and having the media pH initially adjusted to 8. DMT production was not observed in FaNMT-expressing E. coli under these conditions. The 150 mg/L tryptamine supplementation was not exhausted during these studies.
[0143] Promoter Library Screening Under Monitored pH Helps Identify Most Suitable Promoter-. Following the trends observed from the temperature, and pH dependent production of NMT and DMT, a panel of transcriptionally varied mutants was screened to select genetically superior strains for the production of the methylated tryptamines. A promoter library of both INMT and aNMT plasmids were made following the ePathOptimize method (Jones et al., 2015). IPTG inducible promoters from the weakest to strongest: G6, H9, H10, C4, and T7 and constitutive promoters XylA and GAP were all independently tested. Methylated tryptamine production was tested by culturing cells in a media at pH 7.5 and 42 °C with supplemental tryptamine. In order to combat the decrease in pH over time (FIG.
3 A), the pH was readjusted to 7.5 every 2 h over the course of 10 hrs following the pH controlled methods described above and as shown in FIG. 3B. FIGs. 5A and 5B show the NMT and DMT concentrations observed in the INMT and aNMT promoter library, respectively. For both INMT and aNMT mediated methylations, use of the T7 promoter yielded the highest concentrations of both NMT and DMT. NMT and DMT production from INMT under the control of the T7 consensus promoter reached titers of 1.79 +/- 0.37 mg/L and 0.14 +/- 0.03 mg/L, respectively (FIG. 5A). aNMT production of NMT and DMT were also highest under T7 consensus facilitated expression with titers reaching 1.64 +/- 0.20 mg/L and 0.02 +/- 0.01 mg/L, respectively (FIG. 5B). These results led to the use of the T7 promoter for all further experimentation with both INMT and PaNMT. [0144] Strict pH Monitoring Increases NMT and DMT Production'. With the results from the temperature, pH, and genetic optimization assays for methylated tryptamine production completed, production was scaled up through the use of a bioreactor, which allowed working with greater volumes as well as allow us to maintain a constant pH throughout the fermentation (FIG. 3C). BL21 Star™ (DE3) w/ pETM6-SDM2x-INMT (T7 promoter) was used as a model bioreactor strain due to its consistently higher concentrations of both NMT and DMT as compared to aNMT in previous well plate studies. Informed by previously pH-controlled production of NMT and DMT, production was scaled up through the use of pH stat controlled bioreactor fermentations. NMT, DMT, and TMT production titers were observed, at pH 6.5, 7.0, 7.5, and 8.0. FIG. 6 shows the concentrations of NMT, DMT, and TMT as a function of pH. All concentrations represented in FIG. 6 are from bioreactor samples taken 24 h after inoculation. For all three methylated tryptamine compounds of interest (NMT, DMT and TMT), fermentations conducted under pH-stat conditions of pH = 7.5 yielded the highest titers. NMT titers from pH 7.5 fermentations reached 11.44 +/- 2.01 mg/L, a 1.5-fold increase over highest NMT titers observed from 48 well plate assays. DMT titers from pH 7.5 fermentations reached 12.73 +/- 3.28 mg/L, which mark a 7.8-fold increase compared to DMT produced from the top performing 48 well plate assays. TMT titers were also observed in the largest quantities from the pH 7.5 fermentations at a value of 6.76 +/- 2.52 mg/L representing the first TMT production observed from a bacterial culture.
[0145] Extended Pathways Towards the de novo Methylation of Tryptamines'. The synthesis of DMT was further explored by inducing the expression of PsiD, the enzyme found in Psilocybe cubensis responsible for decarboxylating tryptophan to produce tryptamine, alongside the expression of INMT/ aNMT. This dual expression made it possible to produce NMT from growth media supplemented with either tryptophan, serine and indole, or glucose as visualized in FIG. 2.
[0146] Further studies, including promoter library optimization, were conducted in tryptophan supplemented media to reduce metabolic burden and show proof of concept of the newly constructed metabolic pathway. All promoter library screens were performed using the pH controlled, medium throughput screening assay described previously. Lead strains from the promoter library screens were selected for their ability to produce the highest titers of DMT from tryptophan. The selected strains were then used to test the viability of de novo DMT biosynthesis. FIGs. 9A-9D illustrate the success of the promoter library screening in identifying a pathway construct capable of producing more DMT from tryptophan as compared to the T7-INMT-PsiD expressing strain. The T7-INMT-PsiD was created as an initial proof of concept for de novo production of DMT and has been labeled to easily compare its performance within the promoter library screening results. FIG. 9A shows the combined results of two separate promoter library screens with varied operon gene orientation, specifically, xx5-PsiD-INMT and xx5-INMT-PsiD. The number of strains screened is ranged between 5-10 times the library size. A total of 96 strains were selected and are represented in Figure 9A, with 48 strains selected per operon gene orientation. The ‘xx5’ notation indicates the pseudorandom incorporation of 5 mutant T7 promoters of varied strength and determines the library size and the number of strains that need to be screened to account for representation of all possible promoter-gene combinations. Given the large number of strains that need to be screened for promoter library representation, strains were tested in singlicate (n =1), and lead mutants were later isolated and rescreened in replicate. FIG. 9B represents the monocistronic library screen of 144 strains for the gene orientation INMT-PsiD, and it should be noted that a monocistronic promoter library with the gene construct PsiD-INMT could not be created due to limitations in plasmid construction methods. Within the operon, monocistronic, and pseudooperon (xx5-psiD-xx5-INMT) promoter libraries, the best performing strains resulted in just under 25 mg/L DMT and NMT (FIGs. 9A-9C) with the highest TMT titer just under 12mg/L observed within the monocistronic promoter library (FIG. 9B). The pseudooperon library screening results represented in FIG. 9D (144 strains screened) reveal that the INMT-psiD gene expression order was less successful compared to the pseudooperon library screening represented in FIG. 9C (144 strains screened) with a psiD-INMT gene expression order and was the least successful overall in providing high DMT producing strains compared to the others.
[0147] FIG. 10 shows the comparison in DMT production between top to middle performing strains selected from each promoter library screening presented in FIGs. 9A-9D. Strains represented in FIG. 10 were labeled and identified according to the promoter library they were selected from, and the number that was assigned during initial screening: M = monocistronic, P = pseudooperon, O = operon. T7 -INMT-PsiD was again used as a baseline comparison for the success of the promoter library in increasing DMT titers. All strains, with the exception of M29, 020, and Ml 32 produced significantly more DMT than T7-INMT- PsiD (p < 0.05). Strains Ml 11, M21, Pl 17, P29 and O1 were selected for all future methylation screenings to present a relatively broad range of methylation activity. Selected strains were also sent for sequencing yielding the following promoter identities: Ml 11 = H9- INMT-G6-PsiD, M21 = H10-INMT-G6-PsiD, Pl 17 = G6-PsiD-C4-INMT, P29 = HlO-PsiD- C4-INMT, and O1 = C4-PsiD-INMT.
[0148] These top strains were also tested for their ability to catalyze de novo biosynthesis (FIG. 11). Screening conditions were conserved from previous studies; however, tryptophan was not supplemented into the media, such that glucose represents the sole carbon source for growth and product formation. All selected promoter library strains produced significantly more DMT (p < 0.05) than the T7-INMT-PsiD strain, with the best strain producing 14 +/- 0.37 mg/L DMT and 31.3 +/- 0.84 mg/L of total methylated tryptamines (FIG. 11).
[0149] Discussion'. This study shows the first reported case of in vivo NMT, DMT, and TMT production using a prokaryotic host. Additionally, this study demonstrates the effectiveness of genetic, and fermentation conditions optimization for the purpose of enhancing desired product titers.
[0150] Through the use of 48 well plate assays to test a large number of fermentation parameters, it was possible to identify key components to successful chemical product synthesis. HPLC-MS analysis aided in the identification of desired products within the culture media confirming the presence of NMT, DMT, and TMT and thus confirming the hypothesis that INMT and 7’aNMT could be used to enable in vivo methylation of tryptamine in E. coli.
[0151] Identifying the optimal pH and temperature for INMT and PaNMT methylation activity led to a large increase in product titers over traditional E. coli batch fermentation conditions of 37 °C at a neutral initial pH that is not strictly controlled. Furthermore, by employing stricter limits on pH fluctuation over time, final titers of NMT and DMT were seen to increase compared to non-pH controlled cultures. Consistent trends were observed among INMT methylation activity with respect to temperature and pH; however, aNMT activity remained unpredictable due to low activity, with NMT and DMT concentrations frequently near the limit of quantification. The FaNMT-containing E. coli strain was less successful in producing both NMT and DMT compared to the strain containing INMT, under their respective optimal conditions. Furthermore, in vivo results described herein show aNMT activity decreasing at increased fermentation temperature, which was not expected as aNMT activity was previously shown to increase with temperature in an in vitro system (Morris et al. 2018).
[0152] Genetic optimization through the testing of a wide range of different strength promoters, including both inducible and constitutive expression, demonstrated the importance of the strongly inducible T7 promoter in the production of NMT, DMT, and TMT. Library construction and screening of transcriptional variants ensured the most productive pathway configuration for the production of products of interest had been identified. Without wishing to be bound by theory, although the initial screening demonstrated a clear preference for the T7 promoter, extended pathways may be rescreened via the pH-controlled medium throughput well plate assay demonstrated above. As the biosynthesis pathway is extended, the metabolic burden and associated co-factor and precursor needs from the native E. coli metabolism will change, directly affecting the interplay between expression level for the exogenous pathway and resource availability for endogenous metabolism (Wu et al. 2016).
[0153] Scaled-up production to bench top fermenters proved successful as INMT methylation of tryptamine was seen to increase as observed through enhanced titers of NMT, DMT, and the first observation of TMT biosynthesis. The only variables monitored and controlled through the use of benchtop fermenters were the pH, maintained at 7.5, and the temperature, controlled at 42 °C. Dissolved oxygen (DO) was not monitored. Additionally, the fed-batch functionality for both carbon and substrate feed was not utilized. Had either the DO been monitored, or substrate continuously fed to the fermenter, it is expected that product titers would increase beyond levels observed in this study. It should also be addressed that the TMT identified in the bioreactor studies presented above has not been well described in terms of its potential psychoactive effects, and has only minimal mention in the peer reviewed literature (Servillo et al. 2012). Due to the structural similarity to the natural product, aeruginascin, it is expected that TMT may have significant psychoactive activity motivating further study to enhance its production and exploration of its pharmacological potential in animal studies. Furthermore, since the biosynthesis of NMT, DMT, and most notably, TMT, was observed to be catalyzed by the human INMT, this indicates that these mono- and trimethylated derivatives may play a currently unstudied role in human health. The development of an E. coZz-based process to facilitate the efficient biosynthesis of these compounds can lead to more focused studies to determine the roles and mechanism of actions for tryptamines in human neurobiology.
[0154] The de novo biosynthesis of DMT was attempted in this study to eliminate the need for relatively more expensive substrates such as tryptophan, tryptamine, serine, or indole, compared with D-glucose, a cheaper and more readily available substrate.
[0155] Through the expression of transcriptionally-varied genetic mutants of PsiD and INMT onto a single plasmid construct using the same ePathOptimize approach (Jones et al., 2015) we used for the single INMT/PaNMT gene constructs, we were able to achieve de novo production of NMT and DMT in vivo (FIG. 11). A total of six gene constructs were selected from an iterative screening process of each library to be further tested for their ability to produce DMT from either tryptophan or glucose. Final data analysis led to the identification of the top de novo performing strain Ml 11, which contains a monocistronic gene construct with the low strength T7 mutant promoters H9 and G6 controlling the expression of INMT and PsiD, respectively (H9-INMT-G6-PsiD). Through screening of genetically-varied libraries, we were able to demonstrate the first example of a microbe capable of de novo (from glucose) synthesis of NMT, DMT, and TMT in a bacterial host platform.
[0156] Through the studies outlined herewith it has been shown that DMT and DMT derivatives can be synthesized in vivo,- however, the bioreactor fermentation studies indicate that limitations in this biosynthetic production platform of DMT exist. The presence of unmethylated tryptamine in the LCMS analysis corroborates the idea that INMT methylation of tryptamine is the rate limiting step for the in vivo biosynthesis of DMT and DMT derivatives described in this work. UB et al., 2014, suggest that the activity of INMT is attenuated by both non-competitive and competitive inhibition of DMT on the methyltransferase. We believe that if DMT could be removed from the media as it was produced, this potential inhibition of INMT could be reduced or eliminated. To test this hypothesis, we screened two potential hydrophobic overlays as a potential sink for DMT during fermentation. Unfortunately, we observed minimal partition of methylated tryptamines into either hydrophobic overlay, limiting our ability to reduce any INMT inhibition that may be present. More thorough study using a more diverse range of hydrophobic overlays may provide valuable insight into an appropriate path forward towards enhanced fermentationbased production of methylated tryptamines.
[0157] In vivo production of DMT using genetically engineered E. coli has been demonstrated herewith. The data clearly articulates the ability for E. coli to produce DMT. Thus far, de novo production of NMT is the result of a genetically engineered plasmid expressing genes through the use of a pseudo-operon transcriptional configuration. Past research involving the in vivo production of psilocybin, a tryptamine derivative, has shown that, when expressing multiple genes on a single plasmid, an operon format is responsible for highest product yields (Adams et al. 2019). Moving forward, it is necessary to test and compare the production success of an operon gene configuration to that of the pseudo-operon configuration presented in this study. Furthermore, expansion to monocistronic or mixed transcriptional configurations could lead to further enhanced production.
[0158] Additionally, it is important to highlight the success of the preliminary scale-up performed in this study. Moving from simple 48 well plate assays described above, to bench- top fermenters, marked a remarkable 7.5-fold increase in DMT titers. Taking into account the lack of any form of dissolved oxygen monitoring and control, and the lack of either a carbon source or substrate feed, it is evident there is capacity for additional improvement. Further fermentation optimization in this area will undoubtedly result in further enhanced DMT titers as demonstrated in previous work for the in vivo production of psilocybin and is paramount to increased product titer, rate, and yield (Adams et al., 2019).
[0159] Additionally, we consider the possibility of optimizing the de novo production of DMT by leveraging multiple metabolic pathways necessary to the process. By targeting specific pathways essential to the production of DMT, such as SAM regeneration and tryptophan biosynthesis, we believe we can push the metabolic flux towards DMT.
[0160] Activity of these in vivo A-methylation platforms on a variety of alternative tryptamine substrates is evaluated. These non-natural substrates are created through the documented promiscuity towards various commercially available indole derivatives of the native tryptophan synthase, TrpB, to make non-natural tryptophan analogs followed by in vivo decarboxylation using the psilocybin pathway enzyme, PsiD. Both of these enzymes have wide substrate preference and high documented activity in a bacterial host platform. A list of commercially available indole derivatives is provided in Table 2.
[0161] In conclusion, the biosynthesis of DMT through application of metabolic and pathway engineering principles in E. coli presented in this work is the first instance of in vivo and de novo DMT synthesis in a prokaryotic host. DMT titers of about 38 mg/L from tryptophan were obtained. Further efforts to genetically optimize the de novo DMT synthesis pathway, and to tailor the fermentation process to enhance DMT production could lead to an alternative production method competitive with the chemical synthesis of DMT (Cozzi & Daley, 2020; Speeter & Anthony, 1954). As regulations around the studies of DMT and its derivatives for medical use continue to relax moving forward, it is important that avenues for the supply and discovery of these medical compounds are continuously explored.
Table 1
Figure imgf000043_0001
Figure imgf000044_0001
Table 2. List of Commercially Available Indole Derivatives
[0162] Indole Derivatives in the table are available from Fisher Scientific, Massachusetts, USA or from Avantor, Pennsylvania, USA.
Figure imgf000044_0002
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Example 2; Bioreactor Fermentation with Glucose Feed Increases DMT Titers
[0163] Benchtop bioreactor fermentation was carried out similarly to previously described bioreactor methods but with the addition of a glucose feed, which previously went unused as initial studies utilized a pH-controlled batch operation paradigm. Glucose concentrations within the fermentation media was monitored using methods outlined above. With the addition of glucose fed batch strategy, we aimed to revisit the viability of DMT production scale-up; as a result, we chose to ferment our T7-INMT strain with tryptamine supplementation at both 37 °C and 42 °C to compare directly to previous bioreactor data (FIG. 6). Additionally, we wished to compare the DMT production outputs of the T7-INMT strain with tryptamine supplementation to a strain that contained both INMT and PsiD with tryptophan supplementation, in this case strain Mi l l. FIG. 12 shows the end point NMT, DMT and TMT titers observed under a glucose fed batch condition. Strain Mi l l was grown in AMM supplemented with 1 g/L of tryptophan. Strain T7-INMT was grown in AMM supplemented with 150 mg/L tryptamine. Although T7-INMT produced more DMT when fermented at 42°C compared to 37°C it was not significant (p > 0.05), FIG. 12. On the other hand, the addition of the glucose feed made a significant difference in DMT titers compared to previous bioreactor batch fermentations (FIG. 6), showing a 6-fold increase from 12.7 mg/L to 80.1 mg/L. (p < 0.05). Furthermore, Ml 11, was able to produce more NMT and DMT from tryptophan than the T7-INMT strain from tryptamine under fed-batch conditions, suggesting the methyltransferase step to be rate limiting.
[0164] The highest reproducible titers of DMT achieved in this study is reported as 80.1 mg/L, a total of a 48-fold increase from our first proof of concept well plate assays, which was synthesized by co-expression of the human INMT enzyme and the Psilocybe cubensis PsiD gene and produced from tryptophan supplemented media at a maximum molar yield of 0.050 mol/mol.
Example 3: Implementation of Hydrophobic Overlays Fail to Resolve INMT Inhibition
[0165] Before testing overlays on cell cultures, we tested the efficacy of both dodecane and DINP for their ability to extract DMT from media. MS chromatographs show that less than 1% of the DMT partitioned into dodecane after a 72 h incubation period with media containing DMT at relevant levels for the biosynthetic system presented herein. Only 15% of the DMT partitioned into DINP when incubated for 24 h. Despite the lack of observed efficacy, DINP was used as an overlay during a 50mL flask fermentation. We did not observe an increase in DMT production between cultures with an overlay compared to those without. We determined the effect the overlay had on cell viability by comparing ODeoo values between cultures with and without overlays. ODeoo readings of cultures with an overlay were not significantly different (p > 0.05) than those without and overlay.
Example 4; Production of 5-MeO-DMT and Bufotenine
Standard Curves
[0166] 5-MeO-DMT and Bufotenine standard curves were created via serial dilutions of 1.0 mg/mL pure reagent stock solutions purchased from Cerilliant and Lipomed respectively. The slope of the linear portion of the Mass Spectroscopy Extracted Ion Chromatograph (MS-EIC) peak area was used to quantify respective product concentrations.
Results
[0167] Native E. coli TrpB, and heterologous PsiD and INMT were shown to exhibit substrate promiscuity through the production of the 5-MeO-DMT, and bufotenine (5- hydroxy-7V,7V-dimethyltryptamine) via the metabolic pathway described in FIG. 2. 5-MeO- DMT and bufotenine are both psychoactive DMT derivatives that reside in some species of plants and the Colorado River Toad, found in Sonoran Desert. These compounds have recently seen increased use recreationally and spiritually and have anecdotally been known to help treat mental health conditions (Davis et al. 2019). The same elite performing strains isolated for the de novo production of DMT were used for this screening process. Results led to the identification of strain Pl 17, which contains a pseudo-operon gene construct with the weak G6 promoter controlling the expression of PsiD and the strong C4 promoter controlling INMT expression (G6-PsiD-C4-INMT), as the most effective strain in producing both 5- MeO-DMT and bufotenine from their respective indole precursors (FIG. 2). After producing DMT derivatives we believed that we could further utilize the observed substrate promiscuity in attempt to produce psilocin (4-HO-DMT), the active form of the native mushroom psychedelic psilocybin, by feeding the substrate 4-HO-indole using the same pathway previously described. Unfortunately, after comparing screening results to a standard, we were unable to detect any presence of psilocin in our fermentation media. The substrate promiscuity demonstrated by the production of these DMT analogs suggests that additional DMT derivatives, both existing and novel, could be produced through this metabolic pathway.
[0168] By leveraging the substrate promiscuity of both PsiD and INMT in tandem with E. coli 's native tryptophan synthase subunit TrpB, we demonstrated that our production platform could process indole derivatives 5-methoxyindole (5-MeO-indole) and 5- hydroxyindole (5-HO-indole) into DMT derivatives 5-MeO-DMT and Bufotenine, respectively, following the metabolic pathway from FIG. 2. Strains used during this screening were the same used for the de novo production of DMT. The pH-controlled, medium throughput screening method was used to realize the production of both of these DMT derivatives. FIG. 13 shows the production of 5-MeO-NMT and 5-MeO-DMT by select strains with maximum observed titers of 0.67 +/- 0.02 mg/L and 0.23 +/- 0.02 mg/L, respectively, by strain Pl 17. FIG. 14 shows the production of 5-HO-methylatedtryptamines by select strains with a maximum observed titer of 2.64 +/- 0.003 mg/L, 3.58 +/- 0.02 mg/L, and 0.39 +/- 0.05 mg/L of 5-HO-NMT, Bufotenine, and 5-HO-TMT, respectively, also by strain Pl 17.
Example 5; Indoles used for the production of tryptophan and tryptamine products
[0169] A preliminary set of indoles focused on functional group substitutions at the 4, 5, and 6 position were evaluated in an in vivo E. coli model expressing TrpB (endogenous to E. coli host), psiD, psiK, and/or psiM, with the latter three being expressed from a plasmid based expression platform. (See, WO2021/086513, international application no. PCT/US20/051543, which is hereby incorporated by reference in its entirety and for all purposes). These studies demonstrated some level of promiscuous activity towards the majority of indoles tested as verified by the formation of the expected enzymatic reaction product, as detected by liquid chromatography mass spectroscopy analysis. Table 3 provides all indoles from the preliminary set that demonstrated some formation of enzymatic reaction products.
Table 3. Indoles with Documented Activity In Vivo
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Bibliography
[0170] WHO Depression and Other Common Mental Disorders. (2017). WHO.
[0171] Adams, Alexandra M. et al. 2022. “Development of an E. Coli-Based Norbaeocystin Production Platform and Evaluation of Behavioral Effects in Rats.” Metabolic Engineering Communications 14: e00196.
[0172] Adams, A. M., Kaplan, N. A., Wei, Z., Brinton, J. D., Monnier, C. S., Enacopol, A. L., Ramelot, T. A., & Jones, J. A. (2019). In vivo production of psilocybin in E. coli. Metabolic Engineering, 56, 111-119.
[0173] Anderson, B. T., Labate, B. C., Meyer, M., Tupper, K. W., Barbosa, P. C. R., Grob, C. S., Dawson, A., & McKenna, D. (2012). Statement on ayahuasca. In International Journal of Drug Policy (Vol. 23, Issue 3, pp. 173-175).
[0174] Axehod, J. (1961). Enzymatic formation of psychotomimetic metabolites from normally occurring compounds. Science, 134(3415), 343.
[0175] Barbosa, P. C. R., Strassman, R. J., da Silveira, D. X., Areco, K., Hoy, R., Pommy, J., Thoma, R., & Bogenschutz, M. (2016). Psychological and neuropsychological assessment of regular hoasca users. Comprehensive Psychiatry, 71, 95-105.
[0176] Bouso, J. C., Gonzalez, D., Fondevila, S., Cutchet, M., Fernandez, X., Ribeiro Barbosa, P. C., Alcazar-Corcoles, M. A., Araujo, W. S., Barbanoj, M. J., Fabregas, J. M., & Riba, J. (2012). Personality, psychopathology, life attitudes and neuropsychological performance among ritual users of ayahuasca: A longitudinal study. PLoS ONE, 7(8), 42421. [0177] Cozzi, N. V., & Daley, P. F. (2020). Synthesis and characterization of high-purity N,N-dimethyltryptamine hemifumarate for human clinical trials. Drug Testing and Analysis, 12( ), 1483-1493. [0178] Davis, Alan K. et al. 2019. “5-Methoxy-N,N-Dimethyltryptamine (5-MeO-DMT) Used in a Naturalistic Group Setting Is Associated with Unintended Improvements in Depression and Anxiety.” The American journal of drug and alcohol abuse 45(2): 161. /pmc/articles/PMC6430661/ (June 26, 2022).
[0179] de Osorio, F. L., Sanches, R. F., Macedo, L. R., dos Santos, R. G., Maia-De-Oliveira, J. P., Wichert-Ana, L., de Araujo, D. B., Riba, J., Crippa, J. A., & Hallak, J. E. (2015).
Antidepressant effects of a single dose of ayahuasca in patients with recurrent depression: A preliminary report. Revista Brasileira de Psiquiatria, 37(1), 13-20.
[0180] Englaender, Jacob A. et al. 2017. “Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. Coli.” ACS Synthetic Biology 6(4): 710-20.
[0181] Fava, M. (2003). Diagnosis and definition of treatment-resistant depression. In Biological Psychiatry (Vol. 53, Issue 8, pp. 649-659). Elsevier USA.
[0182] Forsstrbm, T., J. Tuominen, and J. Karkkainen. 2001. “Determination of Potentially Hallucinogenic N-Dimethylated Indoleamines in Human Urine by HPLC/ESI-MS-MS.” Scandinavian Journal of Clinical and Laboratory Investigation, 61(7), 547-556.
[0183] Fricke, J., Blei, F., & Hoffmeister, D. (2017). Enzymatic Synthesis ofPsilocybin. Angewandte Chemie - International Edition, 56(40), 12352-12355.
[0184] Fricke, J., Lenz, C., Wick, J., Blei, F., & Hoffmeister, D. (2019). Production Options for Psilocybin: Making of the Magic. Chemistry - A European Journal, 25(4), 897-903.
[0185] Guimaraes dos Santos, R. (2013). Safety and Side Effects of Ayahuasca in Humans- An Overview Focusing on Developmental Toxicology. Journal of Psychoactive Drugs, 45(f), 68-78.
[0186] He, W., Fu, L., Li, G., Andrew Jones, J., Linhardt, R. J., & Koffas, M. (2015).
Production of chondroitin in metabolically engineered E. coli. Metabolic Engineering, 27, 92-100.
[0187] Hoefgen, S., Lin, J., Fricke, J., Stroe, M. C., Mattern, D. J., Kufs, J. E., Hortschansky, P., Brakhage, A. A., Hoffmeister, D., & Valiante, V. (2018). Facile assembly and fluorescence-based screening method for heterologous expression of biosynthetic pathways in fungi. Metabolic Engineering, 48, 44—51.
[0188] Jang, Hui Jeong et al. 2011. “Retinoid Production Using Metabolically Engineered Escherichia Coli with a Two-Phase Culture System.” Microbial Cell Factories 10. [0189] Jones, J. A., Vemacchio, V. R., Lachance, D. M., Lebovich, M., Fu, L., Shirke, A. N., Schultz, V. L., Cress, B., Linhardt, R. J., & Koffas, M. A. G. (2015). EPathOptimize: A combinatorial approach for transcriptional balancing of metabolic pathways. Scientific Reports, 5.
[0190] Knoth, R. L., Bolge, S. C., Kim, E., & Tran, Q.-V. (2010). Effect of inadequate response to treatment in patients with depression. The American Journal of Managed Care, 16(8), el88-196.
[0191] Lima, O. G. (1946). Observances sobre o “Vinho da Jurema” utilizado pelos indios Pancaru de Tacaratu (Pernambuco). Arquivos Do Institute de Pesquisas Agrondmicas, 4, 45- 86.
[0192] Mandell, A. J., & Morgan, M. (1971). Indole(ethyl)amine N-methyltransferase in human brain. Nature New Biology, 230(11), 85-87.
[0193] Manske, R. H. F. (1931). A SYNTHESIS OF THE METHYLTRYPTAMINES AND SOME DERIVATIVES. Canadian Journal of Research, 5(5), 592-600.
[0194] Miller, M. J., Albarracin-Jordan, J., Moore, C., & Capriles, J. M. (2019). Chemical evidence for the use of multiple psychotropic plants in a 1 ,000-year-old ritual bundle from South America. Proceedings of the National Academy of Sciences of the United States of America, 166(23), 11207-11212.
[0195] Milne, N., Thomsen, P., Molgaard Knudsen, N., Rubaszka, P., Kristensen, M., & Borodina, I. (2020). Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives. Metabolic Engineering, 60, 25- 36.
[0196] Morris, J. S., Groves, R. A., Hagel, J. M., & Facchini, P. J. (2018). An n- methyltransferase from ephedra sinica catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production. Journal of Biological Chemistry, 293(35), 13364-13376.
[0197] Pacher, P., & Kecskemeti, V. (2005). Trends in the Development of New Antidepressants. Is there a Light at the End of the Tunnel? Current Medicinal Chemistry, 11(1), 925-943.
[0198] Penn, E., & Tracy, D. K. (2012). The drugs don’t work? antidepressants and the current and future pharmacological management of depression. Therapeutic Advances in Psychopharmacology, 2(5), 179-188. [0199] Saavedra, J. M., Coyle, J. T., & Axelrod, J. (1973). The distribution and properties of the nonspecific N-methyltransferase in brain. Journal of Neurochemistry, 20(3), 743-752.
[0200] Servillo, L., Giovane, A., Balestrieri, M. L., Cautela, D., & Castaldo, D. (2012). N- methylated tryptamine derivatives in citrus genus plants: Identification of N, N, N - trimethyltryptamine in bergamot. Journal of Agricultural and Food Chemistry, 60(37), 9512- 9518.
[0201] Shen, Hong-Wu, Xi-Ling Jiang, Jerrold C. Winter, and Ai-Ming Yu. 2010.
“Psychedelic 5-Methoxy-N,N-Dimethyltryptamine: Metabolism, Pharmacokinetics, Drug Interactions, and Pharmacological Actions.” Current drug metabolism 11(8): 659.
[0202] Souza, Rita C.Z. et al. 2019. “Validation of an Analytical Method for the Determination of the Main Ayahuasca Active Compounds and Application to Real Ayahuasca Samples from Brazil.” Journal of Chromatogr.
[0203] Speeter, M. E., & Anthony, W. C. (1954). Furochromones and Coumarins. XI. the Molluscicidal Activity of Bergapten, Isopimpinillin and Xanthotoxin. Journal of the American Chemical Society, 76(23), 6208-6210.
[0204] Szara, S. (1956). Dimethyltryptamin: Its metabolism in man; the relation of its psychotic effect to the serotonin metabolism. Experientia, 12( ), 441—442.
[0205] Thompson, M. A., Moon, E., Kim, U. J., Xu, J., Siciliano, M. J., & Weinshilboum, R.
M. (1999). Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization. Genomics, 61(3), 285-297.
[0206] UB, Chu et al. 2014. “Noncompetitive Inhibition of Indolethylamine-N-
Methyltransferase by N,N-Dimethyltryptamine and N,N-Dimethylaminopropyltryptamine.” Biochemistry 53(18): 2956-65.
[0207] Wu, G., Yan, Q., Jones, J. A., Tang, Y. J., Fong, S. S., & Koffas, M. A. G. (2016). Metabolic Burden: Cornerstones in Synthetic Biology and Metabolic Engineering Applications. In Trends in Biotechnology (Vol. 34, Issue 8, pp. 652-664). Elsevier Ltd.
[0208] Xu, P., Vansiri, A., Bhan, N., & Koffas, M. A. G. (2012). EPathBrick: A synthetic biology platform for engineering metabolic pathways in E. coli. ACS Synthetic Biology, 1(7), 256-266.
[0209] Young, Kevin P. et al. 2021. “Health Care Workers’ Mental Health and Quality of Life during COVID- 19: Results from a Mid-Pandemic, National Survey.” Psychiatric Services 72(2): 122-28. Table 4; Sequences
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
* * *
[0210] 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 a methylated tryptamine or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, PoNMT, INMT, and combinations thereof; and culturing the host cell.
2. The method of claim 1, wherein the methylated tryptamine is a methylated tryptamine of
Formula I:
Figure imgf000070_0001
wherein:
R1 is selected from the group consisting of NH2, NHCH3, N(CH3)2, N(CH3)3+;
R2 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R3 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R4 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
-68- R5 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R6 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4;
R7 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, COOH, CHO, CN, SO3, SO4, and PO4; and
R8 is selected from the group consisting of H, C1-C5 alkyl, C1-C5 alkoxy, halogen, OH, NO2, NH2, CHO, COOH, CN, SO3, SO4, and PO4.
3. The method of claim 1, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
4. The method of claim 1, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
5. The method of claim 1, wherein the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
6. The method of claim 1, wherein the PaNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
7. The method of claim 1, wherein the INMT gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 60%, at least 70%, at least
-69- 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
8. 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.
9. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, oNMT, INMT, and combinations thereof, all under control of a single promoter in operon configuration.
10. The method of claim 9, 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.
11. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, oNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
12. The method of claim 11, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
13. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
-70-
14. The method of claim 13, 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.
15. The method of claim 1, wherein the intermediate of a methylated tryptamine is an indole or derivatized indole, tryptophan or derivatized tryptophan, tryptamine or derivatized tryptamine.
16. The method of any one of claims 1-15, wherein the host cell is cultured with a supplement independently selected from the group consisting of indole, serine, threonine, methionine and combinations thereof.
17. The method of any one of claims 1-16, wherein the host cell is cultured with a supplement produced by contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, INMT, and combinations thereof; and culturing the host cell in the presence of an indole of Table 2 or Table 3.
18. The method of claim 16 or 17, wherein the supplement is fed continuously to the host cell.
19. The method of any one of claims 1-18, wherein the host cell is grown in an actively growing culture.
20. A recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, zNMT, IMNT, and combinations thereof.
21. The recombinant prokaryotic cell of claim 20, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 14, 22, 32 or a sequence
-71- having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
22. The recombinant prokaryotic cell of claim 20, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 24, 28, 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
23. The recombinant prokaryotic cell of claim 20, wherein the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 20, 26, 30, 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
24. The recombinant prokaryotic cell of claim 20, wherein the aNMT gene encodes a polypeptide comprising the amino acid sequence of 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.
25. The recombinant prokaryotic cell of claim 20, wherein the INMT gene encodes a polypeptide comprising the amino acid sequence of 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.
26. The recombinant prokaryotic cell of claim 20, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glut ami cum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
27. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM,
-72- aNMT, IMNT, and combinations thereof, wherein the one or more methylated tryptamine production genes are under control of a single promoter in operon configuration.
28. The recombinant prokaryotic cell of claim 27, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
29. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, oNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
30. The recombinant prokaryotic cell of claim 29, 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.
31. The recombinant prokaryotic cell of claim 20, wherein the expression vector comprises a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
32. The recombinant prokaryotic cell of claim 31 , 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.
33. An expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, all under control of a single promoter in operon configuration.
-73-
34. The expression vector of claim 33, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
35. A transfection kit comprising the expression vector of claim 33.
36. An expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, aNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in pseudooperon configuration.
37. The expression vector of claim 36, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
38. A transfection kit comprising the expression vector of claim 36.
39. An expression vector comprising a methylated tryptamine production gene selected from the group consisting of psiD, psiK, psiM, zNMT, IMNT, and combinations thereof, wherein each gene is under control of a separate promoter in monocistronic configuration.
40. The expression vector of claim 35, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
41. A transfection kit comprising the expression vector of claim 40.
PCT/US2022/074579 2021-08-05 2022-08-05 Methods for the production of methylated tryptamine derivatives, intermediates or side products WO2023015279A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163203970P 2021-08-05 2021-08-05
US63/203,970 2021-08-05

Publications (1)

Publication Number Publication Date
WO2023015279A1 true WO2023015279A1 (en) 2023-02-09

Family

ID=85156355

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/074579 WO2023015279A1 (en) 2021-08-05 2022-08-05 Methods for the production of methylated tryptamine derivatives, intermediates or side products

Country Status (1)

Country Link
WO (1) WO2023015279A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180216137A1 (en) * 2017-01-26 2018-08-02 Manus Bio, Inc. Metabolic engineering for microbial production of terpenoid products
WO2021067626A2 (en) * 2019-10-01 2021-04-08 Intima Bioscience, Inc. Genetic engineering of fungi to modulate tryptamine expression
US20210108238A1 (en) * 2018-03-08 2021-04-15 New Atlas Biotechnologies Llc Processes for the production of tryptamines
US20210147888A1 (en) * 2019-11-15 2021-05-20 Cb Therapeutics, Inc. Biosynthetic production of psilocybin and related intermediates in recombinant organisms

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180216137A1 (en) * 2017-01-26 2018-08-02 Manus Bio, Inc. Metabolic engineering for microbial production of terpenoid products
US20210108238A1 (en) * 2018-03-08 2021-04-15 New Atlas Biotechnologies Llc Processes for the production of tryptamines
WO2021067626A2 (en) * 2019-10-01 2021-04-08 Intima Bioscience, Inc. Genetic engineering of fungi to modulate tryptamine expression
US20210147888A1 (en) * 2019-11-15 2021-05-20 Cb Therapeutics, Inc. Biosynthetic production of psilocybin and related intermediates in recombinant organisms

Similar Documents

Publication Publication Date Title
US20220372494A1 (en) Methods for the production of psilocybin and intermediates or side products
US10975395B2 (en) Method and cell line for production of polyketides in yeast
Adams et al. In vivo production of psilocybin in E. coli
Yang et al. Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex gene expression knockdown
Germann et al. Glucose‐based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae
Park et al. Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli
Boghigian et al. Multi‐factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions
CN107435049B (en) Recombinant escherichia coli for producing salidroside, construction method and application
BR112017020561B1 (en) MUTANT TRANSAMINASE, FUSION PROTEIN, AMINE PRODUCTION METHOD AND USE OF MUTANT TRANSAMINASE
Davis et al. Nicotiana benthamiana as a transient expression host to produce auxin analogs
Cao et al. Cryptic sulfur incorporation in thioangucycline biosynthesis
Yang et al. Characterization of the xiamenmycin biosynthesis gene cluster in Streptomyces xiamenensis 318
Zhang et al. Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli
Jin et al. Metabolic engineering of Bacillus subtilis 168 for the utilization of arabinose to synthesize the antifungal lipopeptide fengycin
Friedberg et al. In vivo biosynthesis of N, N-dimethyltryptamine, 5-MeO-N, N-dimethyltryptamine, and bufotenine in E. coli
CN110713962A (en) Genetic engineering bacterium for high-yield production of malonyl coenzyme A and construction method and application thereof
WO2023015279A1 (en) Methods for the production of methylated tryptamine derivatives, intermediates or side products
Liu et al. Rational chromosome engineering of Escherichia coli for overproduction of salidroside
WO2022150840A1 (en) Psilocybin and norbaeocystin compositions and methods of treatment
BR112019014116A2 (en) H-ACTIVE S-CYANIDRINE LIASE AND APPLICATION OF THE SAME
WO2022150854A1 (en) Systems and methods for pharmaceutical production of psilocybin and intermediates or side products
WO2023081833A1 (en) Metabolic engineering methods for the production of psilocybin and intermediates or side products
Xiang et al. Total Biosynthesis of Mutaxanthene Unveils a Flavoprotein Monooxygenase Catalyzing Xanthene Ring Formation
Huang et al. Engineering of a Substrate Affinity Reduced S-Adenosyl-methionine Synthetase as a Novel Biosensor for Growth-Coupling Selection of L-Methionine Overproducers
WO2023081837A9 (en) Methods for the production of tryptophans, tryptamines, intermediates, side products and derivatives

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22854114

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE