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WO2023015279A1 - Procédés de production de dérivés de tryptamine méthylée, intermédiaires ou produits secondaires - Google Patents

Procédés de production de dérivés de tryptamine méthylée, intermédiaires ou produits secondaires Download PDF

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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
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mutant
group
promoter
tryptamine
methylated
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John Andrew Jones
Lucas FRIEDBERG
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Miami University
University of Miami
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Miami University
University of Miami
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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.

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Abstract

L'invention concerne des procédés, des cellules hôtes procaryotes, des vecteurs d'expression et des kits pour la production de tryptamine méthylée ou d'un intermédiaire ou d'un produit secondaire de celle-ci. Selon certains modes de réalisation, la cellule hôte procaryote est choisie dans le groupe constitué par Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, et Streptomyces venezuelae.
PCT/US2022/074579 2021-08-05 2022-08-05 Procédés de production de dérivés de tryptamine méthylée, intermédiaires ou produits secondaires Ceased WO2023015279A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP4426818A4 (fr) * 2021-11-05 2025-11-26 Univ Miami Procédés pour la production améliorée de psilocybine et d'intermédiaires ou de produits secondaires par optimisation enzymatique

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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 (fr) * 2019-10-01 2021-04-08 Intima Bioscience, Inc. Génie génétique de champignons pour moduler l'expression de tryptamine
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

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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 (fr) * 2019-10-01 2021-04-08 Intima Bioscience, Inc. Génie génétique de champignons pour moduler l'expression de tryptamine
US20210147888A1 (en) * 2019-11-15 2021-05-20 Cb Therapeutics, Inc. Biosynthetic production of psilocybin and related intermediates in recombinant organisms

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4426818A4 (fr) * 2021-11-05 2025-11-26 Univ Miami Procédés pour la production améliorée de psilocybine et d'intermédiaires ou de produits secondaires par optimisation enzymatique

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