WO2021110992A1 - Non-naturally occurring bacteria modified able to produce tryptophan derived compounds - Google Patents

Non-naturally occurring bacteria modified able to produce tryptophan derived compounds Download PDF

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WO2021110992A1
WO2021110992A1 PCT/EP2020/084773 EP2020084773W WO2021110992A1 WO 2021110992 A1 WO2021110992 A1 WO 2021110992A1 EP 2020084773 W EP2020084773 W EP 2020084773W WO 2021110992 A1 WO2021110992 A1 WO 2021110992A1
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dmt
naturally occurring
nucleic acid
tryptophan
methyltransferase
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PCT/EP2020/084773
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French (fr)
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Henrik SCHOEFER
Patrick Schmitt
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Synbionik Gmbh
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    • 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
    • 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)
    • 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
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01028—Aromatic-L-amino-acid decarboxylase (4.1.1.28), i.e. tryptophane-decarboxylase

Definitions

  • Non-naturally occurring bacteria modified able to produce tryptophan derived compounds.
  • the invention pertains to the field of biotechnology and the genetic modification of microorganisms to produce compounds of substances of interest for the area of medicine, microbiology, pharmacology, genetic engineering, psychiatry, and/or psychology.
  • DMT is a N-methylated derivative of tryptamine carrying two additional methyl groups at the side chain nitrogen atom.
  • N,N-Dimethyltryptamine (DMT) is a naturally occurring chemical substance found in many organisms. Its chemical structure is closely related to the neurotransmitter serotonin and to other psychedelic compounds found in nature e.g. psilocin, the active compound found in “magic mushrooms” (Ratsch, Christian. The encyclopedia of psychoactive plants: ethnopharmacology and its applications. Simon and Schuster, 2005).
  • DMT dimethyltryptamine N-methyltransferase
  • AADC aromatic amino acid decarboxylase
  • 5-HT receptor system serotoninrgic system
  • This receptor system consists of numerous receptor subtypes and it is an essential part of neuronal communication.
  • 5-HT receptors can be found in the central and the peripheral nervous system as well as in other tissues. DMT's action is not limited to the 5-HT receptor system, but also, as recently shown, DMT is the only known endogenous agonist for another type of receptor, the sigma-receptor (Fontanilla,
  • the hallucinogen N, N-dimethyltryptamine is an endogenous sigma- 1 receptor regulator.” Science 323.5916 (2009): 934-937).
  • the sigma-receptor was believed to be an orphan receptor until DMT was found to mediate some of its effects via this receptor.
  • Activation of the sigma-receptor by DMT is associated with observed neural/cell protection correlated to the presence of DMT in the experimental setup (Szabo, Attila, et al.
  • N, N-dimethyltryptamine displays potent protective effects against hypoxia via sigma-1 receptor activation in human primary iPSC-derived cortical neurons and microglia-like immune cells.” Frontiers in neuroscience 10 (2016): 423). In another set of experiments, it was shown that the endogenous concentration of DMT is affected by the simulation of medical incidents, in this case a provoked heart-attack (Dean, Jon G., et al. "Biosynthesis and extracellular concentrations of N, N-dimethyltryptamine (DMT) in Mammalian Brain.” Scientific reports 9.1 (2019): 1-11).
  • DMT also shows a substrate behavior for the serotonin uptake transporter and the vesicular monoamine transporter and therefore is actively transported across membrane barriers (Fontanilla, Anthony, et al. "The hallucinogen N, N- dimethyltryptamine (DMT) is an endogenous sigma- 1 receptor regulator.” Science 323.5916 (2009): 934-937).
  • DMT alone can only be active when given by a parenteral route.
  • repeated injection of DMT in small doses normally not sufficient to produce its primary effects, microdosing has been shown to reduce anxiety and depression (Cameron, Lindsay P., et al. "Chronic, intermittent microdoses of the psychedelic N, N-Dimethyltryptamine (DMT) produce positive effects on mood and anxiety in rodents. "ACS chemical neuroscience 10.7 (2019): 3261- 3270). Due to the work of Rick Strassmann, who conducted FDA approved experiments with healthy probands and injected DMT during the late 1990's, there also is some data available regarding this route of administration and its effects in man (Strassman et al 1995).
  • DMT its endogenous occurrence, the low toxicity, the rapid uptake into the human brain after injection and its role in neural cell survival, make DMT a favorable candidate as a true live saving emergency medication. Since there is no reliable, scalable and cost-effective production method of DMT for the use in a medicinal context, the biotechnological invention seeks to provide an approach for the synthesis of DMT and related compounds in a safe, sustainable and efficient way.
  • the present invention relates generally to methods of production of DMT, as well as expression of enzymes and recombinant microorganisms for the biosynthesis of DMT in particular DMT for use in pharmaceutical composition.
  • the invention is based, in part, on the discovery that the DMT pathway can be efficiently expressed in microorganisms, such as bacteria or yeast, and utilized for production of DMT or DMT derivatives, such as 5-MeOH-N,N-Dimethyltryptamine.
  • the invention also provides methods for expressing the DMT pathway in microorganisms.
  • the present invention relates to a method of production of DMT or DMT derivatives, for example 5-MeOH-N,N-Dimethyltryptamine, in microorganisms, such as Escherichia coli, comprising the heterologous expression of the Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, (AADC)) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 4.1.1.28 and Indolethylamine N-methyltransferase (INMT) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 2.1.1.49.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, (AADC)
  • AADC Aromatic Amino acid Decarboxylase
  • IMT Indolethylamine N-methyltransferase
  • the cassette comprises at least one of the genes encoding one of more enzymes of the DMT pathway, preferably the cassette comprises at least one of the genes encoding Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, (AADC)) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 4.1.1.28 or Indolethylamine N-methyltransferase (INMT) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 2.1.1.49
  • the invention also relates to plasmids and microorganisms used for DMT production.
  • a recombinant expression cassette for producing DMT in a cell the recombinant expression cassette comprising a nucleic acid coding sequence of at least one enzyme of the DMT pathway selected from the group comprising
  • AGCGTAA Indolethylamine N-methyltransferase (INMT) enzyme
  • SEQ ID_NO 3 Indolethylamine N- methyltransferase (INMT)
  • a microorganisms used for DMT production expressing at least one possible isoform of amino acid sequence of the Aromatic Amino acid decarboxylase enzyme (EC Nr.: 4.1.1.28 is identified as SEQ ID_NO 2:
  • the genes encoding Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and Indolethylamine N-methyltransferase (INMT) are on the same expression vector.
  • the invention describes the biosynthetic production of N,N-Dimethyltryptamine (DMT). This is enabled by the overexpression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N-methyltransferase (INMT) (EC Nr. : 2.1.1.49) used to convert tryptamine DMT.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, AADC
  • IMT Indolethylamine N-methyltransferase
  • the nucleic acid coding sequence of Tryptophan Decarboxylase 4 having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 1, or a species homolog thereof, wherein the Tryptophan Decarboxylase retains Tryptophan Decarboxylase activity.
  • the nucleic sequence encoding Tryptophan Decarboxylase has a sequence of SEQ ID NO 1.
  • the nucleic acid coding sequence of Tryptophan Decarboxylase is codon-optimized for expression in a host cell.
  • the Tryptophan Decarboxylase translation and activity can be determined by transcriptome, proteome and metabolome analysis known in the art. In particular, the activity of the Putative Dimethyltryptamine 4-hydroxylase polypeptides or any variants can be tested.
  • the nucleic acid coding sequence of Aromatic Amino acid decarboxylase enzyme encodes a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 2, or a species homolog thereof, wherein the Aromatic Amino acid decarboxylase enzyme retains Tryptamine 4-monooxygenase activity.
  • the Aromatic Amino acid decarboxylase enzyme has a sequence of SEQ ID NO 2.
  • the nucleic acid coding sequence of Aromatic Amino acid decarboxylase enzyme is codon-optimized for expression in a host cell.
  • Aromatic Amino acid decarboxylase enzyme gene transcription and Aromatic Amino acid decarboxylase enzyme translation and activity can be determined by transcriptome, proteome and metabolome analysis known in the art. In particular, the activity of the Aromatic Amino acid decarboxylase enzyme polypeptides or any variants can be tested.
  • the nucleic acid coding sequence of Isoform of Indolethylamine N- methyltransferase (INMT) enzyme encodes a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 4, or a species homolog thereof, wherein the Isoform of Indolethylamine N-methyltransferase (INMT) enzyme polypeptide retains Isoform of Indolethylamine N-methyltransferase (INMT) enzyme activity.
  • the Isoform of Indolethylamine N-methyltransferase (INMT) enzyme polypeptide has a sequence of SEQ ID NO 4.
  • the nucleic acid coding sequence of Isoform of Indolethylamine N-methyltransferase (INMT) enzyme is codon-optimized for expression in a host cell.
  • the Isoform of Indolethylamine N-methyltransferase (INMT) enzyme gene transcription and Isoform of Indolethylamine N-methyltransferase (INMT) enzyme translation and activity can be determined by transcriptome, proteome and metabolome analysis known in the art.
  • the activity of the Isoform of Indolethylamine N-methyltransferase (INMT) enzyme polypeptides or any variants can be tested.
  • the cell and control cell expressing Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N-methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)) further express heterologous Tryptophan hydroxylyase identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 1.14.16.4, Tryptamine-5 -hydroxylyase identified in the Comprehensive Enzyme Information System BRENDA as EC Nr.: 1.14.14.1, and Hydroxyindole-O-Methyltransferase identified in The Comprehensive Enzyme Information System BRENDA as EC-Nr. 2.1.1.4.
  • the genes encoding Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase (SEQ ID NO: 1) or Indolethylamine N-methyltransferase (SEQ ID NO: 3) are operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the modified microorganism/cell.
  • the cell lacks the DMT pathway, and thus lacks the ability to regulate DMT production resulting from the DMT pathway. Accordingly, further provided is a cell comprising the sequences described above, wherein the cell lacks the DMT pathway.
  • the cell is selected from bacteria.
  • the cell is a bacterium, and may be of a genus selected from the genus Escherichia, Saccharomyces, Clostridium, Bacillus, Lactococcus, Zymomonas, Corynebacterium, Pichia, Candida, Hansenula, Trichoderma, Acetobacterium, Ralstonia, Cupravidor, Salmonella, Klebsiella, Paenibacillus, Pseudomonas, Lactobacillus, Rhodococcus, Enterococcus, Alkaligenes, Brevibacterium, Methylobacterium, Methylococcus, Methylomonas, Methylocystis and Methylosinus .
  • the microorganism is selected from the group comprising of Escherichia coli, Saccharomyces cerevisiae, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharbutyricum, Clostridium saccharoperbutylacetonicum,
  • Clostridium buiyricum Clostridium diolis, Clostridium kluyveri, Clostridium pasterianium, Clostridium novyi, Clostridium difficile, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium phytofermentans, Lactococcus lactis, Bacillus subtilis, Bacillus licheniformis, Zymomonas mobilis, Klebsiella oxytoca, Klebsiella pneumonia, Corynebacterium glutamicum, Trichoderma reesei, Ralstonia eutropha, Cupriavidus necator, Pseudomonas putida, Lactobacillus plantarum and Methylob acterium extorquens.
  • the non-naturally occurring microorganism is selected from a group comprising a carboxydotrophic bacteria from the genus Clostridium, Moorella, Oxobacter, Acetobacterium, Eubacterium or Butyribacterium.
  • the invention describes a genetically engineered microorganism used for the biotechnological production of biosynthetic DMT or DMT derivatives, via DMT precursor molecules such as L- Tryptophan, Tryptamine and N-Methyltryptamine (NMT).
  • DMT precursor molecules such as L- Tryptophan, Tryptamine and N-Methyltryptamine (NMT).
  • NMT N-Methyltryptamine
  • the genetically engineered microorganism is selected from the genus Escherichia, Saccharomyces, Clostridium, Bacillus, Lactococcus, Zymomonas, Corynebacterium, Pichia, Candida, Hansenula, Trichoderma, Acetobacterium, Ralstonia, Cupravidor Salmonella, Klebsiella, Paenibacillus, Pseudomonas, Lactobacillus, Rhodococcus, Enterococcus, Alkaligenes, Brevibacterium, Methylobacterium, Methylococcus, Methylomonas, Methylocystis, Methylosinus .
  • the microorganism is selected from the group consisting of E. coli, Saccharomyces cerevisiae, Clostridium acetobutylicum, C beijerinckii, C saccharbutyricum, C. saccharoperbutylacetonicum, C. butyricum, C. diolis, C.
  • the genetically engineered microorganism is selected from a carboxydotrophic bacteria, from the genus Clostridium, Moorella, Oxobacter, Acetobacterium, Eubacterium or Buiyribacterium.
  • the carboxydotrophic microorganism is selected from Clostridium Ijungdahlii, Clostridium carboxydivorans, Clostridium ragsdalei, Clostridium autoethanogenum, Moorella thermoacetica, Moorella thermoautotrophica, Oxobacter pfennigii, Acetobacterium woodi, Eubacterium limosum, Butyribacterium methylotrophicum.
  • the carbon source for the fermentation reaction of genetically engineered carboxydotrophic microorganism is a gaseous substrate containing at least one of CO, C02 and H2.
  • the substrate may be a waste gas obtained as a by-product of an industrial process, or from another source such as from automobile exhaust fumes.
  • the carboxydotrophic microorganism is selected from a group comprising Clostridium Ijungdahlii, Clostridium carboxydivorans, Clostridium ragsdalei, Clostridium autoethanogenum, Moorella thermoacetica, Moorella thermoautotrophica, Oxobacter pfennigii, Acetobacterium woodi, Eubacterium limosum and Butyribacterium methylotrophicum.
  • the microorganism is Escherichia coli BLR (DE3) as it is used for the heterologous expression of unstable proteins or enzymes (Goffin, Philippe, and Philippe Dehottay. "Complete genome sequence of Escherichia coli BLR (DE3), a recA-deficient derivative of E. coli BL21 (DE3).” Genome announcements 5.22 (2017)).
  • the microorganism is Escherichia coli Rosetta 2 (DE3) as it is used for the heterologous expression of eukaryotic proteins or enzymes (Kopanic, Jennifer, et al. "An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain.” Frontiers in pharmacology 4 (2013): 106.).
  • the microorganism is Escherichia coli T7 Express as it is an organism of choice for the general heterologous expression and production of recombinant proteins or enzymes (Lobstein, Julie, et al. "Shuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. "Microbial cell actories 11.1 (2012): 753.).
  • the microorganism is Escherichia coli BL21 (DE3) as it is an organism of choice for the heterologous expression and production of recombinant proteins or enzymes (Rosano, German L., and Eduardo A. Ceccarelli. "Recombinant protein expression in Escherichia coli: advances and challenges.” Frontiers in microbiology 5 (2014): 172.).
  • the microorganism is Zymomonas mobilis ZM4 as it comprises characteristics, for example containing the Entner-Doudoroff pathway, that allow the biosynthetic production of bio-products (e.g. wogonin) more suitable and efficient (He, Ming Xiong, et al. "Zymomonas mobilis: a novel platform for future biorefineries. " Biotechnology for biofuels 7.1 (2014): 101.).
  • bio-products e.g. wogonin
  • the microorganism is Clostridium autoethanogenum as it is an anaerobic microorganism. Therefore, it utilizes an alternative carbon source.
  • the alternative carbon source for the fermentation reaction is a gaseous substrate containing at least one of CO, CO2 and 3 ⁇ 4.
  • the substrate used can come from waste gas obtained as a by-product of an industrial process (for example steel manufacturing, gasification of biomass, coal, animal wastes, production of ferroalloys and municipal solid waste (Wu, Tongwei, et al. "Greatly improving electrochemical N2 reduction over Ti02 nanoparticles by iron doping.
  • the genetically engineered microorganism is optimized for DMT production.
  • the biosynthetic production of DMT or structurally related compounds such as 5-MeOH-DMT or beta-carbolines is based on the aromatic amino acid (AAA) tryptophan. Tryptophan is enzymatically decarboxylated to tryptamine, which serves as a biosynthetic precursor for these substances.
  • AAA aromatic amino acid
  • tryptophan is enzymatically decarboxylated to tryptamine, which serves as a biosynthetic precursor for these substances.
  • 5-MeOH-DMT or beta-carbolines enhanced levels of tryptophan are pursued. This can be achieved by manipulating the endogenous tryptophan biosynthesis pathway of the microorganism at one or more different steps of tryptophan biosynthesis.
  • the microorganism is genetically modified in one or more ways.
  • aroF gene aroG gene or aroH gene
  • D AHP-synthase aroF gene
  • PEP phosphoenolpyruvate
  • erythrose-4- phosphate a condensation reaction between phosphoenolpyruvate (PEP) and erythrose-4- phosphate
  • PEP phosphoenolpyruvate
  • erythrose-4- phosphate mutated by deleting its residue Ilell, making it resistant to feedback inhibition
  • trpED gene encoding for the anthranilate (ANTA) synthase, which catalyzes a biosynthetic step towards tryptophan, is made feedback- resistant by a S40F mutation (Zhao et al. 2011).
  • trpR trp repressor
  • trpR tryptophan degradation enzyme
  • pheA phenylalanine
  • tyrA tyrosine
  • PEP biosynthetic precursor phosphoenolpyruvate
  • pykA pyruvate kinases enzymes
  • pykF pyruvate kinases enzymes
  • csrB negative regulatory RNA, coded by csrB
  • One possible modification involves overexpressing the genes coding for transketolase (tktA) or transaldolase (talB) or the overexpression of the enzyme glucose-6-phosphate dehydrogenase (Rodriguez et al. 2014).
  • the genetically modified microorganism contains one or more genes or gene clusters, which enable the expression of enzymes used for the production of DMT, 5-MeOH-DMT, beta- carbolines from tryptophan and biosynthetic precursors such as tryptamine.
  • the gene or gene clusters contain one or more different combinations of the following enzymes: a. Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC), is involved in decarboxylating tryptophan to tryptamin (EC Nr.: 4.1.1.28).
  • AADC Aromaatic Amino acid Decarboxylase
  • One possible implementation contains the enzyme isoform from Carathanthus roseus (Park et al. 2010), which is codon optimized for the respective microorganism (identified as SEQ ID NO 1):
  • Aromatic Amino acid decarboxylase enzyme (EC Nr. : 4.1.1.28 is identified as SEQ ID_NO 2:
  • EHHVRRVWDLIQKLTDDLLKEA Indolethylamine N-methyltransferase (INMT) is involved in methylating tryptamin (EC Nr.: 2.1.1.49 (formerly EC Nr.: 2.1.1.81)).
  • IMT Indolethylamine N-methyltransferase
  • One possible DNA sequence codes for the human enzyme isoform (Torres et al.
  • the Indolethylamine N-methyltransferase (INMT) enzyme primary sequence can contain one or more mutations in order to optimize the functionality of the protein.
  • the mutations can include one or more of the following mutations: 254C, D28N, H46P, M206V or N245S (Torres et al. 2019)
  • genes or gene cluster combinations can involve one or more genes coding for one or more different isoforms of the enzymes Tryptophan hydroxy lyase (EC Nr.: 1.14.16.4), Tryptamine-5-hydroxylyase (EC Nr.: 1.14.14.1) or Hydroxyindole-O-Methyltransferase (EC-Nr. 2.1.1.4). These genes enable the production of 5-MeOH-DMT.
  • Another possible gene or gene cluster combination involves the introduction of enzymes, which catalyze a Pictet-Sprengler-reaction in order to form 5-Me-OH-DMT from the biosynthetic precursor tryptamine.
  • a recombinant host cell incorporates modifications that increase the intake of precursor, tryptamine, to enable high-titer production of DMT.
  • Preferably sufficient concentrations of tryptamine are present in the culture medium of he microorganism
  • the host cell is modified for enhanced DMT production.
  • a recombinant Escherichia coli cell overexpresses one or more enzymes of the DMT pathway.
  • the host cell is modified for enhanced DMT production.
  • a recombinant Zymomonas mobilis cell overexpresses one or more enzymes of the DMT pathway.
  • the production of DMT is improved by the addition of tryptophan to the media.
  • Tryptophan can be fed at a final concentration of 1 g/1 or at a final concentration of 2 g/1 or at a final concentration of 3 g/1 or at a final concentration of 4 g/1 or at a final concentration of 5 g/1 or at a final concentration of 6 g/1.
  • Tryptophan can be added at the time of inoculation or at 1 h after inoculation or at 2 h after inoculation or at 3 h after inoculation or at 6 h after inoculation or at 9 h after inoculation or at 12 h after inoculation or at 18 h after inoculation or at 24 h after inoculation or at 48 h after inoculation or at 72 h after inoculation.
  • the culturing is carried out at 16-45°C, e.g., room temperature, 37°C, 30- 42 °C, 30-40°C, or 32-38°C.
  • the method comprises recombinantly expressing at least one other member of the DMT pathway in the cell preferably Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N-methyltransferase and culturing the cell in the presence of DMT precursor molecules such as L-Tryptophan, Tryptamine and N-Methyltryptamine (NMT), thereby producing DMT or DMT derivatives in the cell.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, AADC
  • NMT N-Methyltryptamine
  • methods for producing 5-Me-OH-DMT comprises recombinantly expressing at least one other member of the DMT pathway in the cell, and culturing the cell in the presence of DMT precursor molecules such as 5MeO before DMT is formed thereby producing 5-Me-OH-DMT in the cell.
  • the invention also concerns a method of converting a precursor product such as such as L- Tryptophan and/or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT, the method comprising culturing non-naturally occurring microorganism of the invention in a suitable culture medium under conditions suitable to induce expression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N- methyltransferase, and then harvesting the cultured cells or spent medium, thereby converting the precursor product into the target metabolic product such as DMT or 5-Me-OH-DMT.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, AADC
  • AADC Aromatic Amino acid Decarboxylase
  • Indolethylamine N- methyltransferase Indolethylamine N- methyltransferase
  • the method of converting a precursor product such as such as L- Tryptophan and/or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises harvesting and lysing the cultured cells, thereby producing cell lysate. Additionally, the method comprises purifying the target metabolic product i.e. DMT or DMT derivatives such as 5-Me-OH-DMT from the cell lysate, thereby producing a purified target metabolic product.
  • the method of converting a precursor product such as such as L- Tryptophan and/or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises formulating DMT or 5-Me-OH-DMT in a pharmaceutical composition.
  • the method further comprises introducing a recombinant expression cassette to the cell prior to the expressing step.
  • the method further comprises introducing a recombinant expression cassette comprising Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N- methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)), e.g., simultaneously or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the DMT pathway.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, AADC
  • IMT Indolethylamine N- methyltransferase
  • the method further comprises introducing a recombinant expression cassette to the cell prior to the expressing step.
  • the method further comprises introducing a recombinant expression cassette comprising at least Tryptophan hydroxylyase (EC Nr.: 1.14.16.4), Tryptamine-5-hydroxylyase (EC Nr.: 1.14.14.1), and/or Hydroxyindole-O-Methyltransferase (EC-Nr. 2.1.1.4) e.g., simultaneously, or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the 5-MeOH-DMT pathway.
  • the genes or gene clusters which enable the expression of enzymes used for the production of DMT or DMT derivatives, are incorporated into the genetically engineered or enhanced microorganism via a shuttle vector plasmid.
  • the shuttle vector plasmid can contain one or more specific promoters, one or more multiple cloning sites for the insertion of single genes or gene clusters, one or more terminators, one or more resistance cassettes (chosen from AmpR, KanaR, CamR, SpecR, TetR), an E. coli Origin of replication, an Origin of replication specific for the genetically engineered microorganism strain, a T7 IPTG inducible promoter.
  • the shuttle vectors with the incorporated gene cassettes are transformed into the genetically engineered via electroporation and subsequently plated on agar plates containing one or more of the following antibiotics ampicillin, kanamycin, chloramphenicol, tetracycline and spectinomycin. Detection of the successful transformation of gene cassettes is accomplished via PCR and sequencing. The gene transcription and enzyme translation and activity are determined by transcriptome, proteome and metabolome analysis.
  • Figure 1 describes a the process including genetic engineering and chemical molecules involved the invention described.
  • the microorganism can be genetically engineered via the CRISPR-Cas system. This technique enables deletions, insertions or point mutations in the genomic DNA of the microorganism.
  • the gram-negative bacterial strain Zymomonas mobilis is genetically engineered microorganism via the use of the endogenous CRISPR-Cas system.
  • the microorganism is genetically modified via the phage Lambda-derived Red recombination system. This technique enables deletions, insertions or point mutations in the genomic DNA of the microorganism.
  • a plasmid is used comprising the bacteriophage lambda red components.
  • the repair template essential for the homologous recombination is designed to have homologous arms to the genomic DNA of the microorganism where the mutation is incorporated as well as containing the desired mutation. Both the plasmid and the repair template are transformed into the microorganism.
  • the Lambda-derived Red recombination plasmids are transformed by electroporation into the microorganism and subsequently spread on agar plates, supplemented with antibiotics. By colony PCR, the target sequence is amplified with the help of the corresponding primers and individual positive colonies are then identified with Agarose Gel electrophoresis and sequencing.
  • Homologous directed repair is used as a method for the introduction of genetic material to make the non-natural occurring bacterial.
  • the genetic material for the homologous directed repair contains a high degree of homology to the genetic sequence of the microorganism. Through an endogenous in cell mechanism the region with a high degree of homology gets build into the genome creating a non-naturally occurring bacteria.
  • the homologous regions are design in a way that they flank a specific sequence region. This sequence region gets incorporated into the genome of the bacteria through homologous recombination.
  • the incorporated region contains FRT-sequences from Saccharomyces cerevisiae.
  • the FRT sequence is used as a FLP-FRT site-specific recombination system. Gen fragments flanking two FRT sequences are incorporated by the Saccharomyces cerevisiae FLT gen, coding for the FLP recombinase enzyme (Zou et al 2012).
  • Chromatographic methods can include liquid (MeOH, ACN, hexan, water, acetic acid and others) or gaseous mobile phases (3 ⁇ 4, He, N 2 , Ar) and liquid or solid stationary phases including silica gel, polydimethylsiloxane or reversed phase materials.
  • Detection of DMT and related compounds can be achieved by using MS or MS/MS, including Sector mass spectrometry, time-of-flight mass spectrometry, the use of quadrupole mass analyzer, three-dimensional quadrupole ion trap, cylindrical ion trap, linear quadrupole ion trap, orbitrap or fourier transform ion cyclotron resonance and the use of an andequat detector, including electron multiplier systems, faraday cups, ion-to-photon detectors, microchannel plate detectors or inductive detectors.
  • MS or MS/MS including Sector mass spectrometry, time-of-flight mass spectrometry, the use of quadrupole mass analyzer, three-dimensional quadrupole ion trap, cylindrical ion trap, linear quadrupole ion trap, orbitrap or fourier transform ion cyclotron resonance and the use of an andequat detector, including electron multiplier systems, faraday cups, ion-to-photon detectors, microchannel plate detectors
  • UV-absorption, fluorescence, charged aerosol detector, evaporative light scattering detector, flame ionization detector, flame photometric detector, nitrogen phosphorus detector, atomic-emission detector, refractive index detector, radio flow detector, conductivity monitor, thermal conductivity detector, electron capture detector and photoionization detectors or combination of those principles can be applied.
  • DMT can also be detected by using chemical reactions, the use of appropriate stains like iodine vapor, iodoplatinate, marquis reagent, nihydrin, HNCb-atmosphere, NNCD-reagent, PDAB-TS, TACOT, TCBI, vanillin reagents, Van Urk reagent or xanthydrol and the use of an authentic reference substance (Barker et al. 2012; Mulga et al. 2012).
  • Quantification can be achieved by the use of an internal standard containing e.g. 5-Meo-DMT, dr DMT, ds-DMT, 5-Br-DMT or 4F-DMT.
  • an internal standard containing e.g. 5-Meo-DMT, dr DMT, ds-DMT, 5-Br-DMT or 4F-DMT.
  • the products of the fermentation reaction may be recovered from the fermentation broth by continuously removing a portion of the broth from the fermentation bioreactor, separating microbial cells from the broth (by filtration). Then, one or more products can be recovered from the broth simultaneously or sequentially. Preferably returned to the fermentation bioreactor, the cell free permeate remains after the one or more products have been obtained.
  • other non-essential nutrients e.g. vitamins
  • the extraction of tryptamines derivatives from the medium or harvested cells is performed by homogenization of the cells and medium by using mechanical, physical or chemical methods.
  • the extraction of tryptamines derivatives from the medium or harvested cells is not performed homogenization.
  • two-phase extraction method using a non-polar solvent after the basification of the aqueous phase or by a direct extraction from the material using polar or nonpolar solvents is used for DMT enrichment in a first step. Further purification can be achieved by recrystallization or precipitation using a suitable solvent for counter ion system, distillation or sublimation of DMT as well as using typical preparative chromatographic methods.
  • DMT is stored as a salt or as the free base protected from light and oxygen under an inert gas atmosphere, in a solid form or as a solution in water, ethanol, methanol or other solvents.
  • the non-naturally occurring bacteria is grown with 15 N ammonium minimal medium. Enrichment of Tryptamines with isotopes ( 2 H, 3 H, n C, 13 C, 14 C, 13 N, 15 N) can be achieved by the restriction of elemental supply to an enriched source of the corresponding element. Those isotopes modified derivatives can be used for analytical, diagnostical, medicinal or other purposes.
  • the method further comprises introducing the recombinant expression cassette to the cell prior to the expressing step.
  • the method further comprises introducing a recombinant expression cassette comprising Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N- methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)), e.g., simultaneously or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the DMT pathway.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, AADC
  • IMT Indolethylamine N- methyltransferase
  • the method further comprises introducing the recombinant expression cassette to the cell prior to the expressing step.
  • the method further comprises introducing a recombinant expression cassette comprising Tryptophan hydroxylyase (EC Nr.: 1.14.16.4), Tryptamine-5 -hydroxylyase (EC Nr.: 1.14.14.1), and Hydroxyindole-O- Methyltransferase (EC-Nr. 2.1.1.4) e.g., simultaneously, or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the 5- MeOH-DMT pathway.
  • the method further comprises harvesting the DMT emitted from the cell. In some embodiments, the method further comprises harvesting the 5-Me-OH-DMT emitted from the cell.
  • the method results in an increase in the amount of DMT produced by the cell compared to a control cell not recombinant' expressing the at least one member of the DMT pathway.
  • the method results in at least a 2-, 5-, 6-, 8-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 200-, 500-, 600-, 1000-, 1200-, 1600-, 2000-fold or higher fold increase in the amount of DMT produced by the cell compared to a control cell not recombinantly expressing the at least one member of the DMT pathway.
  • the method results in an increase in the amount of 5-Me-OH-DMT produced by the cell compared to a control cell not recombinant' expressing the at least one member of the 5-Me-OH-DMT pathway.
  • the method results in at least a 2-, 3-, 5-, 6-, 8-, 10, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 200-, 500-, 600-, 1000-, 1200-, 1600-, 2000- fold or higher fold increase in the amount of 5-Me-OH-DMT produced by the cell compared to a control cell not recombinantly expressing the at least one member of the 5-Me-OH-DMT pathway.
  • the method results in a cessation of cell growth, i.e., the cells no longer replicate, or grow/ replicate at a greatly reduced rate. In some embodiments, the method results in cell growth 20, 10, 5, 1% or lower compared to a control cell not recombinantly expressing at least one member of the DMT pathway.
  • the precursor molecule, tryptamine is quantitatively converted to DMT, e.g., with an efficiency of 18, 25, 30, 50, 60, 70, 80% or higher.
  • the mass ratio of DMT to dry cell weight is at least 0.25, 0.5, 0.7, 0.8, 0.9, 1 .0 or higher.
  • the DMT -to-biomass (w:w) ratio is at least 0.3%, 0.5%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 1 1.0%, 12.0%, 13.0%, 14.0%, 15.0% or more.
  • the method results in a cessation of cell growth, i.e., the cells no longer replicate, or grow/ replicate at a greatly reduced rate. In some embodiments, the method results in cell growth 20, 10, 5, 1% or lower compared to a control cell not recombinantly expressing at least one member of the 5-Me-OH-DMT pathway. In some embodiments the precursor molecule, tryptamine, is quantitatively converted to 5-Me-OH-DMT, e.g., with an efficiency of 18, 25, 30, 50, 60, 70, 80% or higher.
  • the mass ratio of 5-Me-OH-DMT to dry cell weight is at least 0.25, 0.5, 0.7, 0.8, 0.9, 1 .0 or higher.
  • the DMT -to-biomass (w:w) ratio is at least 0.3%, 0.5%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 1 1.0%, 12.0%, 13.0%, 14.0%, 15.0% or more.
  • the present invention provides methods for making a product comprising DMT, 5-Me-OH-DMT or DMT and 5-Me-OH-DMT.
  • the product is a pharmaceutical composition, a dietary supplement, beverages or a baked good.
  • the DMT and 5-Me-OH-DMT of the present invention can be mixed with one or more excipients to form a pharmaceutical product, which may be a pill, a capsule, a mouth spray, or an oral solution.
  • shuttle vectors with the incorporated genes (Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N- methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)) or Tryptophan hydroxylyase (EC Nr.: 1.14.16.4), Tryptamine-5 -hydroxylyase (EC Nr.: 1.14.14.1)) are transformed into Zymomonas mobilis via electroporation and subsequently plated on agar plates containing one or more of the following antibiotics ampicillin, kanamycin, chloramphenicol, tetracycline and spectinomycin. Detection of the successful transformation of gene cassettes is accomplished via PCR and sequencing. The gene transcription and enzyme translation and activity are determined by transcriptome, proteome and metabolome analysis.
  • a non-naturally occurring microorganism modified to produce N,N-Dimethyltryptamine 2.
  • a non-naturally occurring microorganism according to embodiment 1 expressing the enzymes: a. Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) b. Indolethylamine N-methyltransferase (INMT) (EC Nr. : 2.1.1.49)'
  • Aromatic Amino acid decarboxylase enzyme is a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 2 and wherein the Aromatic Amino acid decarboxylase enzyme retains Tryptamine 4-monooxygenase activity, and b.
  • the Indolethylamine N-methyltransferase (INMT) enzyme is a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 4 and wherein the Indolethylamine N-methyltransferase retains Isoform of Indolethylamine N-methyltransferase (INMT) enzyme activity.
  • a non-naturally occurring microorganism comprising a. a nucleic acid sequence encoding Tryptophan Decarboxylase where in the sequence of the nucleic acid encoding Tryptophan Decarboxylase is at least 80% identical to nucleic acid sequence of SEQ ID No. 1. b. a nucleic acid sequence encoding Indolethylamine N-methyltransferase where in the sequence of the nucleic acid encoding Indolethylamine N-methyltransferase is at least 80% identical to the nucleic acid sequence of SEQ ID No. 3.
  • a non-naturally occurring microorganism according to embodiment 4 comprising an inducible promoter operably linked to the nucleic acid encoding Tryptophan Decarboxylase and/or to the nucleic acid encoding Indolethylamine N-methyltransferase.
  • a non-naturally occurring microorganism according any of embodiments 1-5 modified to produce 5-MeOH- N,N-Dimethyltryptamine 7.
  • microorganism according to any of embodiments 1-7 wherein the microorganism is able to grow in ammonium minimal medium.
  • microorganism is a bacterium or a yeast.
  • Method of converting L-Tryptophan and/or Tryptamine into DMT and/or 5-Me-OH- DMT comprising culturing non-naturally occurring microorganism of any of embodiments 1-12 in a suitable culture medium under conditions suitable to induce expression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N-methyltransferase, and then harvesting the cultured cells or spent medium, thereby converting -Tryptophan and/or Tryptamine into DMT and/or 5- Me-OH-DMT.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, AADC
  • Indolethylamine N-methyltransferase Indolethylamine N-methyltransferase
  • Method of embodiment 13 further comprising the step of : a) lysing the non-naturally occurring microorganism b) purifying DMT and/or 5-Me-OH-DMT from the cell lysate, thereby producing a purified target metabolic product.
  • Method of embodiment 14 further comprising formulating DMT or 5-Me-OH-DMT in a pharmaceutical composition.
  • each step of a metabolic pathway that produces the DMT and 5-Me-OH-DMT, compound of interests occurs in a modified recombinant cell described herein.
  • at least one step of the metabolic pathway occurs in a modified recombinant cell described herein, and at least one step of the metabolic pathway occurs extracellularly, e.g., in microorganism media or within a co-cultured modified recombinant cell.
  • the invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein.
  • any of the terms “comprising”, “consisting essentially of' and “consisting of' may be replaced with either of the other two terms.
  • some embodiments may encompass a host cell “comprising” a number of components, other embodiments would encompass a host cell “consisting essentially of the same components, and still other embodiments would encompass a host cell “consisting of the same components.
  • the terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.
  • nucleic acid sequences provided by the invention is in the form of an expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding polypeptide being involved in DMT and 5-Me-OH-DMT synthesis.
  • the polynucleotides provided by the invention can either be isolated from their natural genomic environment, modified after their isolation, or produced artificially from pure sequence information.
  • genomic DNA is referring to the heritable genetic information of a host organism.
  • Said genomic DNA comprises the entire genetic material of a cell or an organism.
  • genome or genomic DNA refers to the total amount of DNA of a cell, including the DNA of the nucleus (chromosomal DNA), extrachromosomal DNA, and organellar DNA (e.g. of mitochondria).
  • genome or genomic DNA when used in context of eukaryotic organisms, is referring to the chromosomal DNA of the nucleus or genomic DNA or heterologous plasmid DNA.
  • nucleic acid refers to a single or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • the monomer is typically referred to as a nucleotide.
  • Nucleic acids can include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.
  • the terms as used herein, the terms flickabouf ’ andohnaround” indicate a close range around a numerical value when used to modify that specific value. If ‘X” were the value, for example, “about X” or possiblyaround X” would indicate a value from 0.9X to 1.1X, e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or a value from 0.99X to 1.01X.
  • any reference to “about X” or possibly around X” specifically indicates at least the values X, 0.9X, 0.9 IX, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.IX, and values within this range.
  • nucleic acid sequence encoding or a “nucleic acid coding sequence” refers to a nucleic acid which directs the expression of a specific protein or peptide.
  • nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA, and the RNA sequence that is translated into protein.
  • the nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences.
  • the nucleotide sequence is codon-optimized to reflect the typical codon usage of the host cell without altering the polypeptide encoded by the nucleotide sequence.
  • the term "codon optimization” or “codon-optimized” refers to modifying the codon content of a nucleic acid sequence without modifying the sequence of the polypeptide encoded by the nucleic acid to enhance expression in a particular host cell.
  • the term is meant to encompass modifying the codon content of a nucleic acid sequence as a means to control the level of expression of a polypeptide ( e.g. ., either increase or decrease the level of expression).
  • described are nucleic sequences encoding the enzymes involved in the engineered metabolic pathways.
  • a non-naturally occurring microorganism may express one or more polypeptide having an enzymatic activity necessary to perform the steps described below.
  • a particular cell may comprises one, two, three, four, five or more than five nucleic acid sequences, each one encoding the polypeptide(s) necessary to produce DMT or 5-Me-OH- DMT compound, or compound intermediate described herein.
  • a single nucleic acid molecule can encode one, or more than one, polypeptide.
  • a single nucleic acid molecule can contain nucleic acid sequences that encode two, three, four or even five different polypeptides.
  • Nucleic acid sequences useful for the invention described herein may be obtained from a variety of sources such as, for example, amplification of cDNA sequences, DNA libraries, de novo synthesis, excision of genomic segment. The sequences obtained from such sources may then be modified using standard molecular biology and/or recombinant DNA technology to produce nucleic sequences having desired modifications. Exemplary methods for modification of nucleic acid sequences include, for example, site directed mutagenesis, PCR mutagenesis, deletion, insertion, substitution, swapping portions of the sequence using restriction enzymes, optionally in combination with ligation, homologous recombination, site specific recombination or various combination thereof.
  • the nucleic acid sequences may be a synthetic nucleic acid sequence. Synthetic polynucleotide sequences may be produced using a variety of methods described in U.S. Patent No. 7,323,320, as well as U.S. Pat. Appl. Pub. Nos. 2006/0160138 and 2007/0269870.
  • promoter refers to a polynucleotide which directs the transcription of a structural gene to produce mRNA.
  • a promoter is located in the 5' region of a gene, proximal to the start codon of the coding region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent or by the induced release of a suppressor. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter.
  • a polynucleotide is "heterologous to" an organism or a second polynucleotide if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e. g. a genetically engineered coding sequence or an allele from a different ecotype, variety or strain).
  • Transgene refers to a polynucleotide manipulated by man or a copy or complement of a polynucleotide manipulated by man.
  • a trans- genie expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of manipulation by man (e.g., by methods described in Sambrook et al., Molecular Cloning- A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc.
  • a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature.
  • restriction sites or plasmid vector sequences manipulated by man may flank or separate the promoter from the second polynucleotide.
  • polynucleotides can be manipulated in many ways and are not limited to the examples above.
  • the plasmid contains, one or more resistance cassettes (selected from ampicillin resistance (AmpR), kanamycin resistance (KanaR), chloramphenicol resistance (CamR), spectinomycin resistance (SpecR), tetracycline resistance (TetR)) and an Zymomonas mobilis Origin of Replication (OR) or Escherichia coli Origin or Replication (OR).
  • AmpR ampicillin resistance
  • KanaR kanamycin resistance
  • CamR chloramphenicol resistance
  • TelR spectinomycin resistance
  • TetR tetracycline resistance
  • Zymomonas mobilis Origin of Replication OR
  • Escherichia coli Origin or Replication OR
  • the term "recombinant” is used to specify an organism or cell, e.g. a microorganism, it is used to express that the organism or cell comprises at least one "transgene", “transgenic” or “recombinant” polynucleotide, which is usually specified later on.
  • operble linkage or “operably linked” are generally understood as meaning an arrangement in which a genetic control sequence, e.g. a promoter, enhancer or terminator, is capable of exerting its function with regard to a polynucleotide being operably linked to it, for example a polynucleotide encoding a polypeptide.
  • Function in this context, may mean for example control of the expression, i.e.
  • Control in this context, encompasses for example initiating, increasing, governing, or suppressing the expression, i.e. transcription and, if appropriate, translation.
  • Controlling in turn, may be, for example, tissue and / or time specific. It may also be inducible, for example by certain chemicals, stress, pathogens and similar.
  • operable linkage is understood as meaning for example the sequential arrangement of a promoter, of the nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator, in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed.
  • An operably linkage does not necessarily require a direct linkage in the chemical sense.
  • Genetic control sequences such as, for example, enhancer sequences are also capable of exerting their function on the target sequence from positions located at a distance to the polynucleotide, which is operably linked.
  • Preferred arrangements are those in which the nucleic acid sequence to be ex- pressed is positioned after a sequence acting as promoter so that the two sequences are linked covalently to one another.
  • the distance between the promoter and the amino acid sequence encoding polynucleotide in an expression cassette is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
  • an expression cassette may also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for example an endogenous promoter, for example by means of homologous recombination or else by random insertion.
  • Such constructs are likewise understood as being expression cassettes for the purposes of the invention.
  • flanking regions refers to regions or sequences located upstream and/or downstream of a nucleic acid coding sequence in a recombinant expression cassette which is involved in double homologous recombination (e.g., integration) of a portion of the cassette with a host cell's genome.
  • double homologous recombination refers to the ability of nucleic acid sequences to exchange, wherein a nucleic acid stably integrates into the genome of a host cell's DNA sequence to make a new combination of DNA sequence.
  • complementarity refers to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide.
  • sequence A-G-T is complementary to the sequence T-C-A.
  • Complementarity can be partial, in which only some of the nucleic acids match according to base pairing, or complete, where ail the nucleic acids match according to base pairing.
  • protein protein
  • peptide and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid metics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutaniate, and O- phosphoserme.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • the terms "non-naturally occurring amino acid” and "unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • a “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and/or size of the side group chain is maintained.
  • Illustrative sets of amino acids that may be substituted for one another include (i) positively- charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic amino acids Gly, Ala, Val, Leu and He; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro; (viii) small hydroxyl amino acids Ser and Thr; and sulfur-containing amino acids Cys and Met.
  • Reference to the charge of an amino acid in this paragraph refers to the charge at pH 7 0
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Conservatively modified variants can include polymorphic variants, interspecies homologs (orthologs), intraspecies homologs (paralogs), and allelic variants.
  • % identity and its derivatives are used interchangeably herein with the term “% homology” and its derivatives to refer to the level of a nucleic acid or an amino acid sequence’s identity between another nucleic acid sequence or any other polypeptides, or the polypeptide's amino acid sequence, where the sequences are aligned using a sequence alignment program.
  • % homology and its derivatives to refer to the level of a nucleic acid or an amino acid sequence
  • nucleic acids or proteins refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g.
  • sequences are then said to be "substantially identical.”
  • This definition also refers to, and can be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Optimal alignment of such sequences can be carried out by any of the publically available algorithms or programs for determining sequence identity and alignment, e.g., BLAST.
  • GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
  • Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al, BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used.
  • sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motifts), using the programs mentioned above using the default parameters.
  • Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).
  • cassette or "expression cassette” means those constructs in which the nucleic acid sequence encoding an amino acid sequence to be expressed is linked operably to at least one genetic control element which enables or regulates its expression (i.e. transcription and / or translation).
  • An expression cassette typically includes a sequence to be expressed, and sequences necessary for expression of the sequence to be expressed.
  • the sequence to be expressed can be a coding sequence or a non-coding sequence ⁇ e.g., an inhibitory sequence).
  • an expression cassette is inserted into an expression vector (e.g., a plasmid) to be introduced into a host cell.
  • the expression may be, for example, stable or transient, constitutive, or inducible.
  • Expression cassettes may also comprise the coding regions for two or more polypeptides and lead to the transcription of polycistronic RNAs.
  • an RBS in a bacterial e.g., E.
  • cob cell is selected to bind to bacterial (Escherichia cob) ribosomes (e.g., the 16S rRNA), an RBS in a cyanobacterial cell (e.g., Synechocystis) is selected to bind to cyanobacterial ribosomes (e.g., the 16S rRNA) etc.
  • bacterial Erscherichia cob
  • RBS in a cyanobacterial cell
  • cyanobacterial cell e.g., Synechocystis
  • the cell or expression system can be manipulated to include heterologous ribosomes that bind to a particular RBS.
  • transfection and “transformation” refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods.
  • the nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 18.1- 18.88.
  • a polynucleotide or polypeptide sequence is "heterologous to" an organism or a second sequence if it originates from a different species, or, if from the same species, it is modified from its original form.
  • a promoter operability linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
  • a heterologous expression cassette includes sequence!) that are from a different species than the cell into which the expression cassette is introduced, or if from the same species, is genetically modified.
  • Yeast or other eukaryotic species may be introduced on high- level expression plasmid vectors or through genomic integration using methods well known to those skilled in the art. Such methods may involve CRISPR Cas-9 technology, yeast artificial chromosomes (YACs) or the use of retrotransposons. Alternatively, if natural to the host organism, such genes may be up regulated by genetic element integration methods known to those skilled in the art.
  • Recombinant refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism.
  • a recombinant polynucleotide or a copy or complement of a recombinant polynucleotide is one that has been manipulated using well known methods.
  • a recombinant expression cassette comprising a promoter operability linked to a second polynucleotide can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994- 1998)).
  • a recombinant expression cassette (or expression vector) typically comprises polynucleotides in combinations that are not found in nature.
  • recombinant protein is one that is expressed from a recombinant polynucleotide
  • recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).
  • culture when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside its normal environment under controlled conditions, e.g., under conditions suitable for survival.
  • Cultured cells are allowed to survive, and culturing can result in cell growth, stasis, differentiation or division. The term does not imply that all cells in the culture survive, grow, or divide, as some may naturally die or senesce.
  • Cells are typically cultured in media, which can be changed during the course of the culture.
  • media and “culture solution” refer to the cell culture milieu.
  • Media is typically an isotonic solution, and can be liquid, gelatinous, or semi-solid, e.g., to provide a matrix for cell adhesion or support.
  • Media can include the components for nutritional, chemical, and structural support necessary for culturing a cell.
  • media includes a carbon source for biosynthesis and metabolism. In the case of plant or other photosynthetic cell cultures, the carbon source is typically CO2.
  • a "control,” e.g., a control cell, control sample, or control value, refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample or condition.
  • a test sample can include cells exposed to a test condition or a test agent, while the control is not exposed to the test condition or agent (e.g., negative control).
  • the control can also be a positive control, e.g., a known cell exposed to known conditions or agents, for the sake of comparison to the test condition.
  • a positive control can include a cell with a known level of production of the product of interest.
  • a control can also represent an average value gathered from a plurality of samples, e.g., to obtain an average value.
  • a control value can also be obtained from the same cell or population of cells, e.g., from an earlier-obtained sample, prior to the disorder or deficiency, or prior to treatment.
  • controls can be designed for assessment of any number of parameters.
  • One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data.
  • a tryptamine is a monoamine alkaloid, which contains an indole ring structure. It is structurally similar to the amino acid tryptophan from which the name derives. Tryptamine is found in trace amounts in the brains of mammals and is hypothesized to play a role as a neuromodulator or neurotransmitter. Similar to other trace amines tryptamine binds to human trace amine-associated receptor 1 (TAARl) as an agonist. TAAR1 plays a significant role in regulating neurotransmission in dopamine norepinephrine and serotonin neurons in the CNS. Additionally, it also affects immune system and neuroimmune system function through different mechanisms. (Jones RS.
  • Host cells are generally, but not necessarily, cultured in the presence of starting materials, such as tryptamines, hexanoic acid, prenol, isoprenol, or the like, for periods of time ranging from a few hours to a day or longer (e.g., 24 hours, 30 hours, 36 hours, or 48 hours) at temperatures ranging from about 20 °C to about 40 °C depending on the particular host cells employed.
  • starting materials such as tryptamines, hexanoic acid, prenol, isoprenol, or the like
  • starting materials such as tryptamines, hexanoic acid, prenol, isoprenol, or the like
  • temperatures ranging from about 20 °C to about 40 °C depending on the particular host cells employed.
  • host cells transformed or genomically integrated with plasmids or vectors containing at least one or more than one expression cassette.
  • the cells used to produce DMT or 5-Me-OH-DMT as described herein are genetically modified. That is, heterologous nucleic acid is introduced into the cells.
  • the genetically modified cells do not occur in nature.
  • Suitable cells are capable of expressing a nucleic acid construct (expression cassette) encoding biosynthetic enzymes, as described herein.
  • the cell naturally produces at least some biosynthetic precursors, e.g., tryptamine.
  • genes encoding desired enzymes can be heterologous to the cell, or native to the cell but operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the cell.
  • Any microorganism can be used in the present method so long as it remains viable after being transformed with the heterologous genes.
  • Microorganisms used for producing the DMT or 5-Me-OH-DMT e.g., microorganisms lacking the DMT and 5-Me-OH-DMT pathway, e.g., bacteria, cyanobacteria or green microalgae, are engineered to express heterologous enzymes that generate DMT and 5-Me-OH-DMT.
  • nucleic acid constructs described herein can be operably linked to a promoter and/or terminator so that the desired transcript(s) and protein(s) are expressed in a cell cultured under suitable conditions.
  • Methods for designing and making nucleic acid constructs and expression vectors are well known to those skilled in the art.
  • Sequences of nucleic acids encoding the subject enzymes are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning.
  • direct chemical synthesis oligonucleotides of up to about 40 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence.
  • commercial services are available that can supply synthetic genes of the desired sequence.
  • the desired sequences may be isolated from natural sources using well known cloning methodology, e.g., employing PGR to amplify the desired sequences and join the amplified regions.
  • nucleic acid coding sequences for desired biosynthetic enzymes can be incorporated into an expression cassette.
  • Those of ordinary skill in the art are familiar with the necessary steps for incorporating a nucleic acid sequence into an expression cassette, and into an expression vector for introduction to a cell.
  • a typical expression vector contains the desired nucleic acid sequence preceded by one or more regulatory regions (e.g., promoter), along with a ribosome binding site (RBS). Promoters can be either constitutive or inducible, e.g., under certain environmental conditions.
  • the invention is not limited with respect to the precise promoter or expression vector used.
  • any suitable expression vector may be used to incorporate the desired sequences
  • readily available expression vectors include, without limitation: plasmids, such as pET, pGex, pJF119EH, pSClOl, pBR322, pBBRlMCS-3, pUR, EX, pMRIOO, pCR4, pBAD24, pUC 19; and bacteriophages, such as Ml 3 phage and l phage.
  • Certain expression vectors may only be suitable for particular host cells which can be readily determined by one of ordinary skill in the art. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector.
  • Homologous recombination can occur between, the expression vector and the homologous region in one or more genomic copies present in the host cell.
  • a selectable marker present on the expression vector is used to isolate transformant cells having undergone double homologous recombination by a selection method, such as antibiotic resistance or drug resistance.
  • the plasmid contains one or more specific promoters, one or more multiple cloning sites for the insertion of single genes or gene clusters, one or more terminators, one or more resistance cassettes (selected from ampicillin resistance (AmpR), kanamycin resistance (KanaR), chloramphenicol resistance (CamR), spectinomycin resistance (SpecR), tetracycline resistance (TetR)) and an Zymomonas mobilis Origin of Replication (OR) and Escherichia coli Origin of Replication. Detection of the successful transformation of gene cassettes can be accomplished via PCR and sequencing. The gene transcription and enzyme translation and activity are determined by transcriptome, proteome and metabolome analysis.
  • Cell culture techniques are commonly known in the art and described, e.g., in Sambrook, et al. (1989) Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Cells are typically cultured in isotonic media that includes a carbon source, and in some cases, selection factors to select for recombinant cells (e.g., those with antibiotic resistance).
  • the host cell is preferably cultured at a temperature between 22° C and 37° C. While commercial biosynthesis in host cells such as E. coli can be limited by the temperature at which overexpressed and/or foreign enzymes (e.g., enzymes derived from plants) are stable, recombinant enzymes (including the terpenoid synthase) may be engineered to allow for cultures to be maintained at higher temperatures, resulting in higher yields and higher overall productivity.
  • foreign enzymes e.g., enzymes derived from plants
  • recombinant enzymes including the terpenoid synthase
  • the host cell (bacterial or yeast host cell) is cultured at about 22° C or greater, about 23° C or greater, about 24° C or greater, about 25° C or greater, about 26° C or greater, about 27° C or greater, about 28° C or greater, about 29° C or greater, about 30° C or greater, about 31° C or greater, about 32° C or greater, about 33° C or greater, about 34° C or greater, about 35° C or greater, about 36° C or greater, or about 37° C.
  • DMT and 5-Me-OH-DMT can be extracted from media and/or whole cells and recovered.
  • the DMT or 5-Me-OH-DMT are recovered and optionally purified by fractionation (e.g. fractional distillation, chromatography, etc.).
  • the product can be recovered by any suitable process, including partitioning the desired product into an organic phase.
  • the production of the desired product can be determined and/or quantified, for example, by gas chromatography (e.g., GC) or high-pressure liquid chromatography (HPLC-)-.in combination with a suitable detection method, preferably mass spectrometry (MS) or other systems (FID, RI, DAD)
  • MS mass spectrometry
  • FID fluorescence RI
  • DAD field-de
  • the desired product can be produced in batch or continuous bioreactor systems.
  • the amounts of DMT and 5-Me-OH-DMT can be measured in a recombinant host cell to identify rate limiting steps in the biosynthetic pathway. Once a rate-limiting step has been identified, expression or activity of the (limiting) one or more enzyme can be modified by various methods known in the art, such as codon optimization, use of a stronger or weaker promotor, expressing multiple copies of the corresponding gene, and constructing variants with increase stability and/or activity or to knock-out existing genes that might prevent high yield or production.
  • Identification and quantification of DMT and 5-Me-OH-DMT can be performed by several methods combining chromatographic separation; for example LC, HPLC, UHPLC or GC, and mass sensitive or photo optical detection.
  • Chromatographic methods can include liquid (MeOH, ACN, hexan, water, acetic acid and others) or gaseous mobile phases (3 ⁇ 4, He, N 2 , Ar) and liquid or solid stationary phases including silica gel, polydimethylsiloxane or reversed phase materials.
  • Detection of DMT and related compounds can be achieved by using MS or MS/MS including sector mass spectrometry, time-of-flight mass spectrometry, the use of quadrupole mass analyzer, three-dimensional quadrupole ion trap, cylindrical ion trap, linear quadrupole ion trap, orbitrap or fourier transform ion cyclotron resonance and the use of an andequat detector, including electron multiplier systems, faraday cups, ion-to-photon detectors, microchannel plate detectors or inductive detectors.
  • OH-DMT can also be detected by using chemical reactions, the use of appropriate stains like iodine vapor, iodoplatinate, marquis reagent, nihydrin, HN0 3 -atmosphere, NNCD-reagent, PDAB-TS, TACOT, TCBI, vanillin reagents, Van Urk reagent or xanthydrol and the use of an authentic reference substance (Barker et al. 2012 DOI 10.1002/dta.422) (Mulga et al. 2007 ISBN 0-9770876-5-4).
  • Quantification can be achieved by the use of an internal or external standard containing DMT and 5-Me-OH-DMT or isotope labeled derivatives. By the addition of a known amount of these substances prior to sample preparation, it is possible to calculate the actual amount of DMT and 5-Me-OH-DMT and related compounds in each sample (Barker et al. 2013
  • DMT and 5-Me-OH-DMT are produced simultaneously produced by a recombinant host cell are retained within the recombinant cell. DMT and 5-Me-OH-DMT can be recovered from the culture medium or from the recombinant host cell.
  • the invention also concerns a method of converting L-Tryptophan or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT, the method comprising culturing non- naturally occurring microorganism of the invention in a suitable culture medium under conditions suitable to induce expression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N-methyltransferase, and then harvesting the cultured cells or spent medium, thereby converting the precursor product into the target metabolic product.
  • Tryptophan Decarboxylase Aromatic Amino acid Decarboxylase, AADC
  • AADC Aromatic Amino acid Decarboxylase
  • Indolethylamine N-methyltransferase Indolethylamine N-methyltransferase
  • the method of converting a precursor product such as such as L-Tryptophan, Tryptamine and N-Methyltryptamine (NMT) into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises harvesting and lysing the cultured cells, thereby producing cell lysate. Additionally, the method comprises purifying the target metabolic product i.e. DMT or DMT derivatives such as 5-Me-OH-DMT from the cell lysate, thereby producing a purified target metabolic product.
  • a precursor product such as such as L-Tryptophan, Tryptamine and N-Methyltryptamine (NMT) into a target metabolic product
  • NMT N-Methyltryptamine
  • the method of converting a precursor product such as such as L- Tryptophan, Tryptamine and N-Methyltryptamine (NMT) into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises formulating DMT or 5-Me-OH-DMT in a pharmaceutical composition.
  • a non-naturally occurring microorganism modified to produce N,N-Dimethyltryptamine 2.
  • a non-naturally occurring microorganism according to embodiment 1 modified to comprise the enzymes: a. Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) b. Indolethylamine N-methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81))
  • a non-naturally occurring microorganism according to embodiment 1 modified to produce 5-MeOH- N,N-Dimethyltryptamine
  • a non-naturally occurring microorganism according to embodiment 3 comprising one or more genes coding for one or more different isoforms of: a. Tryptophan hydroxylyase (EC Nr.: 1.14.16.4) b. Tryptamine-5-hydroxylyase (EC Nr. : 1.14.14.1), and c. Hydroxyindole-O-Methyltransferase (EC-Nr. 2.1.1.4)
  • microorganism according to embodiment 1 wherein the microorganism is able to grow in ammonium minimal medium.
  • microorganism according to embodiment 3 wherein the microorganism is able to produce N,N-Dimethyltryptamine from ammonium minimal medium.
  • microorganism according to embodiment 8 wherein the microorganism is a bacterium.
  • Figure 1 Presentation of the process including genetic engineering and chemical molecules involved in the invention as described.
  • Figure 1 describes the concept of the invention: A biosynthetic system made of a modified cell which is modified with a vector to comprise the required sequences to acquire the substrates (e.g. Tryptophan) present in the medium and produce DMT.
  • substrates e.g. Tryptophan
  • Figure 2 FC/MS/MS chromatogram showing tryptophan (peak at 4.5 min) in samples prepared from a DMT producing, modified microorganism.
  • Figure 3 FC/MS/MS chromatogram showing tryptamine (peak at 5.2 min) in samples prepared from a DMT producing, modified microorganism.
  • Figure 4 FC/MS/MS chromatogram showing N-methyltryptamine (peak at 5.9 min) in samples prepared from a DMT producing, modified microorganism.
  • Figure 5 FC/MS/MS chromatogram showing N,N-dimethyltryptamine (peak at 6.1 min) in samples prepared from a DMT producing, modified microorganism.
  • Figure 6 FC/MS/MS chromatogram showing tryptophan (peak at 4.5 min) in a sample prepared from commercial tryptophan reference material.
  • Figure 7 FC/MS/MS chromatogram showing tryptamine (peak at 5.2 min) in samples prepared from commercial tryptamine reference material.
  • Figure 8 LC/MS/MS chromatogram showing N-methyltryptamine (peak at 5.9 min) in samples prepared from samples prepared from commercial NMT reference material.
  • Figure 9 LC/MS/MS chromatogram showing N,N-dimethyltryptamine (peak at 6.1 min) in samples prepared from commercial DMT reference material.
  • Figure 11 Signal for DMT when injecting increasing concentrations of DMT reference material.
  • Figure 12 Signal for NMT over the time of the cultivation.
  • Figure 13 Signal for DMT over the time of the cultivation.
  • Figure 14 Signal for tryptophan over the time of the cultivation.
  • Asp Gly lie Glu Arg Val Asp Ser Leu Ser Leu Ser Pro His Lys Trp 305 310 315 320 Leu Leu Ala Tyr Leu Asp Cys Thr Cys Leu Trp Val Lys Gin Pro His 325 330 335
  • Val Leu Asp Ala Gly Phe Asp lie Glu Gin Leu Leu His Ser Pro Gin 225 230 235 240 Ser Tyr Ser Val Thr Asn Ala Ala Asn Asn Gly Val Cys Phe lie Val 245 250 255

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Abstract

The invention describes a non-naturally occurring bacteria modified genetically to produce tryptophan derived compounds including N,N-Dimethyltryptamine. The modified bacteria is preferably Escherichia coli or Zymomonas mobilis and it is modified to express the one or more Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) enzyme, Indolethylamine N-methyltransferase (INMT) enzyme, N-methyltransferase (INMT) enzyme, Tryptophan hydroxylyase, Tryptamine-5-hydroxylyase enzyme, and Hydroxyindole-O-Methyltransferase enzyme.

Description

Non-naturally occurring bacteria modified able to produce tryptophan derived compounds.
Field of the Invention The invention pertains to the field of biotechnology and the genetic modification of microorganisms to produce compounds of substances of interest for the area of medicine, microbiology, pharmacology, genetic engineering, psychiatry, and/or psychology.
Background The use of psychedelic substances (from plant and fungal sources) for medical, recreational and religious purposes has been recorded since ancient cultures (Ratsch et al. 2005). This field has become prominent in the late century due to scientific curiosity and the promising pharmacological properties of these substances (Nichols et al. 2016). One of the largest groups, from a chemical perspective, of naturally occurring hallucinogens belongs to the class of tryptamines. Lysergic acid diethylamide (LSD), a member of the tryptamine family was introduced into the mainstream western medicine as a substance able to aid psychiatrists and their patients with the process of healing conditions such as depression, post-traumatic stress disorder (PTSD), anxiety and other afflictions to the mind (Hofmann, Albert. LSD: my problem child. Oxford University Press, 2013). Eventually, due to its consumption by a healthy population for recreational purposes, LSD was perceived as a harmful threat to moral and order (Hofmann et al. 2013). As a consequence, LSD along with other hallucinogenic substances became criminalized and its use was perceived as taboo. After being placed in the most restrictive category of narcotics by the respective international authorities, its usage in the scientific field became illegal and all ongoing research came to an end. Eventually, most substances related to hallucinogenic effects (such as N,N-Dimethyltryptamine (DMT) and related compounds) became stigmatized causing the number of scientific investigation in the area of psychedelics and their pharmacological properties to decrease. DMT is a N-methylated derivative of tryptamine carrying two additional methyl groups at the side chain nitrogen atom. N,N-Dimethyltryptamine (DMT) is a naturally occurring chemical substance found in many organisms. Its chemical structure is closely related to the neurotransmitter serotonin and to other psychedelic compounds found in nature e.g. psilocin, the active compound found in “magic mushrooms” (Ratsch, Christian. The encyclopedia of psychoactive plants: ethnopharmacology and its applications. Simon and Schuster, 2005). Most commonly, it is either synthesized through the Speeter- Anthony synthesis from indole using oxalyl chloride, dimethylamine, and lithium aluminium hydride as reagents or extracted from numerous plant found in tropical to subtropical areas around the world (Shulgin, Alexander, and Arm Shulgin. TIHKAL: the continuation. Transform press, 1997).
Most recently, this molecule has been found to be undoubtedly present naturally in the mammalian brain as it is created in small amounts during normal metabolism (Barker, Steven A., et al. "LC/MS/MS analysis of the endogenous dimethyltryptamine hallucinogens, their precursors, and major metabolites in rat pineal gland microdialysate." Biomedical Chromatography 27.12 (2013): 1690-1700). On the Example 1 of the patent application
EP0836849, it is described how a battery of neurometabolic tests were performed, including the measurement of dimethyltryptamine in patients with Alzheimer seeking for a treatment of a disorder of muscular hypotonia. DMT is formed from tryptamine by the enzyme indolethylamine N-methyltransferase (INMT) ubiquitously present in non-neural tissues. Also, tryptamine is produced enzymatically from the amino acid tryptophan by the enzyme aromatic amino acid decarboxylase (AADC).
In regard to the effects of hallucinogenic substances such as tryptamines in the human brain and its consciousness is mainly mediated by interactions with 5-HT receptor system (serotonergic system) (Nichols, David E. "Psychedelics." Pharmacological reviews 68.2 (2016): 264-355). This receptor system consists of numerous receptor subtypes and it is an essential part of neuronal communication. 5-HT receptors can be found in the central and the peripheral nervous system as well as in other tissues. DMT's action is not limited to the 5-HT receptor system, but also, as recently shown, DMT is the only known endogenous agonist for another type of receptor, the sigma-receptor (Fontanilla,
Dominique, et al. "The hallucinogen N, N-dimethyltryptamine (DMT) is an endogenous sigma- 1 receptor regulator." Science 323.5916 (2009): 934-937). The sigma-receptor was believed to be an orphan receptor until DMT was found to mediate some of its effects via this receptor. Activation of the sigma-receptor by DMT is associated with observed neural/cell protection correlated to the presence of DMT in the experimental setup (Szabo, Attila, et al. "The endogenous hallucinogen and trace amine N, N-dimethyltryptamine (DMT) displays potent protective effects against hypoxia via sigma-1 receptor activation in human primary iPSC-derived cortical neurons and microglia-like immune cells." Frontiers in neuroscience 10 (2016): 423). In another set of experiments, it was shown that the endogenous concentration of DMT is affected by the simulation of medical incidents, in this case a provoked heart-attack (Dean, Jon G., et al. "Biosynthesis and extracellular concentrations of N, N-dimethyltryptamine (DMT) in Mammalian Brain." Scientific reports 9.1 (2019): 1-11). DMT also shows a substrate behavior for the serotonin uptake transporter and the vesicular monoamine transporter and therefore is actively transported across membrane barriers (Fontanilla, Dominique, et al. "The hallucinogen N, N- dimethyltryptamine (DMT) is an endogenous sigma- 1 receptor regulator." Science 323.5916 (2009): 934-937). These findings, in combination with the overlap between the recognized effects of exogenous DMT and the reported impressions during almost fatal situations (Near-Death- Experience) gives a strong hint that endogenous DMT might be produced by the body in order to protect the central nervous system or other organs during harmful conditions (Nichols et al. 2018).
In its traditional and most distributed form of use, the consumption of the Amazonian beverage Ayahuasca, DMT is used in combination with another family of active compounds, the harmala- alkaloids (Dominguez-Clave, Elisabet, et al. "Ayahuasca: pharmacology, neuroscience and therapeutic potential." Brain research bulletin 126 (2016): 89-101). Only by this pharmacological combination, DMT can become orally active. Without blocking the monoamine-oxidase(MAO)- System, orally administration of DMT, even in higher doses, will not produce any noticeable psychological effects. There are many reports of positive effects mediated by the traditional or non-traditional usage of this combination. Positive effects have been observed regarding different psychological malfunctions such as depression, anxiety or substance dependency (Frecska, Ede, Petra Bokor, and Michael Winkelman. "The therapeutic potentials of ayahuasca: possible effects against various diseases of civilization." Frontiers in Pharmacology 7 (2016): 35).
DMT alone can only be active when given by a parenteral route. In a rodent model, repeated injection of DMT (in small doses normally not sufficient to produce its primary effects, microdosing) has been shown to reduce anxiety and depression (Cameron, Lindsay P., et al. "Chronic, intermittent microdoses of the psychedelic N, N-Dimethyltryptamine (DMT) produce positive effects on mood and anxiety in rodents. "ACS chemical neuroscience 10.7 (2019): 3261- 3270). Due to the work of Rick Strassmann, who conducted FDA approved experiments with healthy probands and injected DMT during the late 1990's, there also is some data available regarding this route of administration and its effects in man (Strassman et al 1995). Investigating a dose/response relationship for i.v. DMT as well as setting up a guideline to its save and proper use under clinical conditions, he gives some information and a follow-up on the long term effects on his volunteers. Currently, studies are being developed regarding the altered state of consciousness caused by intravenous N,N-Dimethyltryptamine in order to investigate the effects of DMT on the power spectrum recorded via multivariate EEG (Timmermann, Christopher, et al. "DMT models the near-death experience." Frontiers in psychology 9 (2018): 1424).
All these promising properties of DMT: its endogenous occurrence, the low toxicity, the rapid uptake into the human brain after injection and its role in neural cell survival, make DMT a favorable candidate as a true live saving emergency medication. Since there is no reliable, scalable and cost-effective production method of DMT for the use in a medicinal context, the biotechnological invention seeks to provide an approach for the synthesis of DMT and related compounds in a safe, sustainable and efficient way.
BRIEF SUMMARY OF THE INVENTION
The present invention relates generally to methods of production of DMT, as well as expression of enzymes and recombinant microorganisms for the biosynthesis of DMT in particular DMT for use in pharmaceutical composition.
(Insert claims here)
(Insert sequences here)
The invention is based, in part, on the discovery that the DMT pathway can be efficiently expressed in microorganisms, such as bacteria or yeast, and utilized for production of DMT or DMT derivatives, such as 5-MeOH-N,N-Dimethyltryptamine. The invention also provides methods for expressing the DMT pathway in microorganisms.
More specifically, the present invention relates to a method of production of DMT or DMT derivatives, for example 5-MeOH-N,N-Dimethyltryptamine, in microorganisms, such as Escherichia coli, comprising the heterologous expression of the Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, (AADC)) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 4.1.1.28 and Indolethylamine N-methyltransferase (INMT) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 2.1.1.49.
Provided herein are expression cassette comprising at least one of the genes encoding one of more enzymes of the DMT pathway, preferably the cassette comprises at least one of the genes encoding Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, (AADC)) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 4.1.1.28 or Indolethylamine N-methyltransferase (INMT) identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 2.1.1.49 The invention also relates to plasmids and microorganisms used for DMT production. In some embodiments, provided is a recombinant expression cassette for producing DMT in a cell, the recombinant expression cassette comprising a nucleic acid coding sequence of at least one enzyme of the DMT pathway selected from the group comprising
(Tryptophan Decarboxylase) SEQ ID NO 01 (Tryptophan Decarboxylase): ATGGGCAGCATTGATAGCACCAACGTGGCGATGAGCAACAGCCCGGTGGGCGA
ATTTAAACCGCT GGAAGCGGAAGAATTT CGC A AAC AGGCGC ATCGC AT GGT GG
ATTTTATTGCGGATTATTATAAAAACGTGGAAACCTATCCGGTGCTGAGCGAAG
TGGAACCGGGCTATCTGCGCAAACGCATTCCGGAAACCGCGCCGTATCTGCCG
GAACCGCTGGATGATATTATGAAAGATATTCAGAAAGATATTATTCCGGGCATG
ACCAACTGGATGAGCCCGAACTTTTATGCGTTTTTTCCGGCGACCGTGAGCAGC
GCGGCGTTTCTGGGCGAAATGCTGAGCACCGCGCTGAACAGCGTGGGCTTTACC
TGGGTGAGCAGCCCGGCGGCGACCGAACTGGAAATGATTGTGATGGATTGGCT
GGCGCAGATTCTGAAACTGCCGAAAAGTTTCATGTTTAGCGGCACCGGCGGCG
GCGTGATTCAGAACACCACCAGCGAAAGCATTCTGTGCACCATTATTGCGGCGC
GCGAACGCGCGCTGGAAAAACTGGGCCCGGATAGCATTGGCAAACTGGTGTGC
TATGGCAGCGATCAGACCCATACCATGTTTCCGAAAACCTGCAAACTGGCGGG
CATTTATCCGAACAACATTCGCCTGATTCCGACCACCGTGGAAACCGATTTTGG
CATT AGCCCGCAGGT GCTGCGCAAAATGGTGGAAGATGATGT GGCGGCGGGCT
ATGTGCCGCTGTTTCTGTGCGCGACCCTGGGCACCACCAGCACCACCGCGACCG
ATCCGGTGGATAGCCTGAGCGAAATTGCGAACGAATTTGGCATTTGGATTCATG
TGGATGCGGCGTATGCGGGCAGCGCGTGCATTTGCCCGGAATTTCGCCATTATC
TGGATGGCATTGAACGCGTGGATAGCCTGAGCCTGAGCCCGCATAAATGGCTG
CTGGCGTATCTGGATTGCACCTGCCTGTGGGTGAAACAGCCGCATCTGCTGCTG
CGCGCGCTGACCACCAACCCGGAATATCTGAAGAACAAACAGAGCGATCTGGA
TAAAGTGGTGGATTTTAAAAACTGGCAGATTGCGACCGGCCGCAAATTTCGCA
GCCTGAAACTGTGGCTGATTCTGCGCAGCTATGGCGTGGTGAACCTTCAGAGCC
ATATTCGCAGCGATGTGGCGATGGGCAAAATGTTTGAAGAATGGGTGCGCAGC
GATAGCCGCTTTGAAATTGTGGTGCCGCGCAACTTTAGCCTGGTGTGCTTTCGC
CT GA AACCGGAT GT GAGC AGCCT GC AT GT GGA AGAAGT GAAC AAGA AACTGCT
GGATATGCTGAACAGCACCGGCCGCGTGTATATGACCCATACCATTGTGGGCG
GCATTTATATGCTGCGCCTGGCGGTGGGCAGCAGCCTGACCGAAGAACATCAT
GTGCGCCGCGTGTGGGATCTGATTCAGAAACTGACCGATGATCTGCTGAAAGA
AGCGTAA (Indolethylamine N-methyltransferase (INMT) enzyme) SEQ ID_NO 3 (Indolethylamine N- methyltransferase (INMT)):
ATGAAAGGCGGCTTTACCGGCGGCGATGAATATCAGAAACATTTTCTGCCGCGCGAT
TATCTGGCGACCTATTATAGCTTTGATGGCAGCCCGAGCCCGGAAGCGGAAATGCTG
AAATTTAACCTGGAATGCCTGCATAAAACCTTTGGCCCTGGCGGCCTTCAGGGCGAT
ACCCTGATTGATATTGGCAGCGGCCCGACCATTTATCAGGTGCTGGCGGCGTGCGAT
AGCTTTCAGGATATTACCCTGAGCGATTTTACCGATCGCAACCGCGAAGAACTGGAA
AAATGGCTGAAGAAAGAACCGGGCGCGTATGATTGGACCCCGGCGGTGAAATTTGC
GTGCGAACTGGAAGGCAACAGCGGTCGCTGGGAAGAAAAAGAAGAAAAACTGCGC
GCGGCGGTGAAACGCGTGCTGAAATGCGATGTGCATCTGGGCAACCCGCTGGCGCCT
GCGGT GCTGCCGCT GGCGGATTGCGT GCTGACCCTGCTGGCGATGGA AT GCGCGTGC
TGTAGCCTGGATGCGTATCGCGCGGCGCTGTGCAACCTGGCGAGCCTGCTGAAACCG
GGCGGCCATCTGGTGACCACCGTGACCCTGCGCCTGCCGAGCTATATGGTGGGCAAA
CGCGAATTTAGCTGCGTGGCGCTGGAAAAAGAAGAAGTGGAACAGGCGGTGCTGGA
T GCGGGCTTTGAT ATTGAAC AGCT GCT GC ATAGCCCGC AGAGCTAT AGCGTGACCAA
CGCGGCGAACAACGGCGTGTGCTTTATTGTGGCGCGCAAGAAACCGGGCCCGTAA
In some embodiments, provided is a microorganisms used for DMT production expressing at least one possible isoform of amino acid sequence of the Aromatic Amino acid decarboxylase enzyme (EC Nr.: 4.1.1.28 is identified as SEQ ID_NO 2:
MGSID S TNV AMSN SP V GEFKPLE AEEFRKQ AHRMVDFI AD YYKNVET YPV SEVEPGYL RKRIPETAPYFPEPFDDIMKDIQKDIIPGMTNWMSPNFYAFFPATVSSAAFFGEMFSTAFN S V GFTWV S SPAATEFEMIVMDWFAQIFKFPKSFMF SGTGGGVIQNTTSESIFCTIIA ARER AFEKFGPDSIGKFVCYGSDQTHTMFPKTCKFAGIYPNNIRFIPTTVETDFGISPQVFRKMV EDDVAAGYVPFFFCATFGTTSTTATDPVDSFSEIANEFGIWIHVDAAYAGSACICPEFRH YFDGIERVDSFSFSPHKWFFAYFDCTCFWVKQPHFFFRAFTTNPEYFKNKQSDFDKW DFKNWQIATGRKFRSLKLWLILRSYGVVNLQSHIRSDVAMGKMFEEWVRSDSRFEIVVP RNF SF V CFRFKPD V S SLHVEEVNKKLLDMLN S T GRVYMTHTIV GGI YMLRF AV GS SETE EHHVRRVWDLIQKLTDDLLKEA; and at least one possible isoform of Indolethylamine N-methyltransferase (INMT) enzyme (EC Nr.: 2.1.1.49 (formerly EC Nr . : 2.1.1.81 )) : MKGGFT GGDEY QKHFLPRD YLATYY SFDGSPSPEAEMLKFNLECLHKTF GPGGLQGDTL IDIGSGPTIYQVLAACDSFQDITLSDFTDRNREELEKWLKKEPGAYDWTPAVKFACELEG NSGRWEEKEEKLRAAVKRVLKCDVHLGNPLAPAVLPLADCVLTLLAMECACCSLDAYR AALCNLASLLKPGGHLVTTVTLRLPSYMVGKREFSCVALEKEEVEQAVLDAGFDIEQLL HSPQSYSVTNAANNGV CFIVARKKPGP
In a preferred embodiment (like in figure 1), the genes encoding Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and Indolethylamine N-methyltransferase (INMT) are on the same expression vector.
The invention describes the biosynthetic production of N,N-Dimethyltryptamine (DMT). This is enabled by the overexpression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N-methyltransferase (INMT) (EC Nr. : 2.1.1.49) used to convert tryptamine DMT.
In some embodiments, the nucleic acid coding sequence of Tryptophan Decarboxylase 4 having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 1, or a species homolog thereof, wherein the Tryptophan Decarboxylase retains Tryptophan Decarboxylase activity. In some embodiments, the nucleic sequence encoding Tryptophan Decarboxylase has a sequence of SEQ ID NO 1. In some embodiments, the nucleic acid coding sequence of Tryptophan Decarboxylase is codon-optimized for expression in a host cell. The Tryptophan Decarboxylase translation and activity can be determined by transcriptome, proteome and metabolome analysis known in the art. In particular, the activity of the Putative Dimethyltryptamine 4-hydroxylase polypeptides or any variants can be tested.
In some embodiments, the nucleic acid coding sequence of Aromatic Amino acid decarboxylase enzyme encodes a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 2, or a species homolog thereof, wherein the Aromatic Amino acid decarboxylase enzyme retains Tryptamine 4-monooxygenase activity. In some embodiments, the Aromatic Amino acid decarboxylase enzyme has a sequence of SEQ ID NO 2. In some embodiments, the nucleic acid coding sequence of Aromatic Amino acid decarboxylase enzyme is codon-optimized for expression in a host cell. The Aromatic Amino acid decarboxylase enzyme gene transcription and Aromatic Amino acid decarboxylase enzyme translation and activity can be determined by transcriptome, proteome and metabolome analysis known in the art. In particular, the activity of the Aromatic Amino acid decarboxylase enzyme polypeptides or any variants can be tested.
In some embodiments, the nucleic acid coding sequence of Isoform of Indolethylamine N- methyltransferase (INMT) enzyme encodes a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 4, or a species homolog thereof, wherein the Isoform of Indolethylamine N-methyltransferase (INMT) enzyme polypeptide retains Isoform of Indolethylamine N-methyltransferase (INMT) enzyme activity. In some embodiments, the Isoform of Indolethylamine N-methyltransferase (INMT) enzyme polypeptide has a sequence of SEQ ID NO 4. In some embodiments, the nucleic acid coding sequence of Isoform of Indolethylamine N-methyltransferase (INMT) enzyme is codon-optimized for expression in a host cell. The Isoform of Indolethylamine N-methyltransferase (INMT) enzyme gene transcription and Isoform of Indolethylamine N-methyltransferase (INMT) enzyme translation and activity can be determined by transcriptome, proteome and metabolome analysis known in the art. In particular, the activity of the Isoform of Indolethylamine N-methyltransferase (INMT) enzyme polypeptides or any variants can be tested. In some embodiments, the cell and control cell expressing Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N-methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)) further express heterologous Tryptophan hydroxylyase identified in The Comprehensive Enzyme Information System BRENDA as EC Nr.: 1.14.16.4, Tryptamine-5 -hydroxylyase identified in the Comprehensive Enzyme Information System BRENDA as EC Nr.: 1.14.14.1, and Hydroxyindole-O-Methyltransferase identified in The Comprehensive Enzyme Information System BRENDA as EC-Nr. 2.1.1.4.
In some embodiments, the genes encoding Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase (SEQ ID NO: 1) or Indolethylamine N-methyltransferase (SEQ ID NO: 3) are operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the modified microorganism/cell. In some embodiments, the cell lacks the DMT pathway, and thus lacks the ability to regulate DMT production resulting from the DMT pathway. Accordingly, further provided is a cell comprising the sequences described above, wherein the cell lacks the DMT pathway. In some embodiments, the cell is selected from bacteria.
In various embodiments, the cell is a bacterium, and may be of a genus selected from the genus Escherichia, Saccharomyces, Clostridium, Bacillus, Lactococcus, Zymomonas, Corynebacterium, Pichia, Candida, Hansenula, Trichoderma, Acetobacterium, Ralstonia, Cupravidor, Salmonella, Klebsiella, Paenibacillus, Pseudomonas, Lactobacillus, Rhodococcus, Enterococcus, Alkaligenes, Brevibacterium, Methylobacterium, Methylococcus, Methylomonas, Methylocystis and Methylosinus .
In a preferred embodiment, the microorganism is selected from the group comprising of Escherichia coli, Saccharomyces cerevisiae, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharbutyricum, Clostridium saccharoperbutylacetonicum,
Clostridium buiyricum, Clostridium diolis, Clostridium kluyveri, Clostridium pasterianium, Clostridium novyi, Clostridium difficile, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium phytofermentans, Lactococcus lactis, Bacillus subtilis, Bacillus licheniformis, Zymomonas mobilis, Klebsiella oxytoca, Klebsiella pneumonia, Corynebacterium glutamicum, Trichoderma reesei, Ralstonia eutropha, Cupriavidus necator, Pseudomonas putida, Lactobacillus plantarum and Methylob acterium extorquens.
In another embodiment the non-naturally occurring microorganism is selected from a group comprising a carboxydotrophic bacteria from the genus Clostridium, Moorella, Oxobacter, Acetobacterium, Eubacterium or Butyribacterium.
The invention describes a genetically engineered microorganism used for the biotechnological production of biosynthetic DMT or DMT derivatives, via DMT precursor molecules such as L- Tryptophan, Tryptamine and N-Methyltryptamine (NMT). The microorganism is genetically engineered in a way that it comprises exogenous genetic material coding for the enzymatic pathway of DMT. In an embodiment the genetically engineered microorganism is selected from the genus Escherichia, Saccharomyces, Clostridium, Bacillus, Lactococcus, Zymomonas, Corynebacterium, Pichia, Candida, Hansenula, Trichoderma, Acetobacterium, Ralstonia, Cupravidor Salmonella, Klebsiella, Paenibacillus, Pseudomonas, Lactobacillus, Rhodococcus, Enterococcus, Alkaligenes, Brevibacterium, Methylobacterium, Methylococcus, Methylomonas, Methylocystis, Methylosinus . In a particular embodiment, the microorganism is selected from the group consisting of E. coli, Saccharomyces cerevisiae, Clostridium acetobutylicum, C beijerinckii, C saccharbutyricum, C. saccharoperbutylacetonicum, C. butyricum, C. diolis, C. kluyveri, C pasterianium, C novyi, C difficile, C thermocellum, C cellulolyticum, C cellulovorans, C phytofermentans, Lactococcus lactis, Bacillus subtilis, Bacillus licheniformis, Zymomonas mobilis, Klebsiella oxytoca, Klebsiella pneumonia, Corynebacterium glutamicum, Trichoderma reesei, Ralstonia eutropha, Cupriavidus necator Pseudomonas putida, Lactobacillus plantarum, Methylob acterium extorquens. In an embodiment the genetically engineered microorganism is selected from a carboxydotrophic bacteria, from the genus Clostridium, Moorella, Oxobacter, Acetobacterium, Eubacterium or Buiyribacterium. In a particular embodiment the carboxydotrophic microorganism is selected from Clostridium Ijungdahlii, Clostridium carboxydivorans, Clostridium ragsdalei, Clostridium autoethanogenum, Moorella thermoacetica, Moorella thermoautotrophica, Oxobacter pfennigii, Acetobacterium woodi, Eubacterium limosum, Butyribacterium methylotrophicum. The carbon source for the fermentation reaction of genetically engineered carboxydotrophic microorganism is a gaseous substrate containing at least one of CO, C02 and H2. The substrate may be a waste gas obtained as a by-product of an industrial process, or from another source such as from automobile exhaust fumes.
In a preferred embodiment the carboxydotrophic microorganism is selected from a group comprising Clostridium Ijungdahlii, Clostridium carboxydivorans, Clostridium ragsdalei, Clostridium autoethanogenum, Moorella thermoacetica, Moorella thermoautotrophica, Oxobacter pfennigii, Acetobacterium woodi, Eubacterium limosum and Butyribacterium methylotrophicum.
In a preferred embodiment the microorganism is Escherichia coli BLR (DE3) as it is used for the heterologous expression of unstable proteins or enzymes (Goffin, Philippe, and Philippe Dehottay. "Complete genome sequence of Escherichia coli BLR (DE3), a recA-deficient derivative of E. coli BL21 (DE3)." Genome announcements 5.22 (2017)). In a preferred embodiment the microorganism is Escherichia coli Rosetta 2 (DE3) as it is used for the heterologous expression of eukaryotic proteins or enzymes (Kopanic, Jennifer, et al. "An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain." Frontiers in pharmacology 4 (2013): 106.).
In a preferred embodiment the microorganism is Escherichia coli T7 Express as it is an organism of choice for the general heterologous expression and production of recombinant proteins or enzymes (Lobstein, Julie, et al. "Shuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. "Microbial cell actories 11.1 (2012): 753.).
In a preferred embodiment the microorganism is Escherichia coli BL21 (DE3) as it is an organism of choice for the heterologous expression and production of recombinant proteins or enzymes (Rosano, German L., and Eduardo A. Ceccarelli. "Recombinant protein expression in Escherichia coli: advances and challenges." Frontiers in microbiology 5 (2014): 172.).
In a preferred embodiment the microorganism is Zymomonas mobilis ZM4 as it comprises characteristics, for example containing the Entner-Doudoroff pathway, that allow the biosynthetic production of bio-products (e.g. wogonin) more suitable and efficient (He, Ming Xiong, et al. "Zymomonas mobilis: a novel platform for future biorefineries. " Biotechnology for biofuels 7.1 (2014): 101.).
In a preferred embodiment the microorganism is Clostridium autoethanogenum as it is an anaerobic microorganism. Therefore, it utilizes an alternative carbon source. The alternative carbon source for the fermentation reaction is a gaseous substrate containing at least one of CO, CO2 and ¾. The substrate used can come from waste gas obtained as a by-product of an industrial process (for example steel manufacturing, gasification of biomass, coal, animal wastes, production of ferroalloys and municipal solid waste (Wu, Tongwei, et al. "Greatly improving electrochemical N2 reduction over Ti02 nanoparticles by iron doping. " Angewandte Chemie International Edition 58.51 (2019): 18449-18453.)), or another source such as from automobile exhaust fumes (Xu Xu, Dan, et al. "Bacterial community and nitrate removal by simultaneous heterotrophic and autotrophic denitrification in a bioelectrochemically-assisted constructed wetland." Bioresource technology 245 (2017): 993-999.).
In an embodiment the genetically engineered microorganism is optimized for DMT production. The biosynthetic production of DMT or structurally related compounds such as 5-MeOH-DMT or beta-carbolines is based on the aromatic amino acid (AAA) tryptophan. Tryptophan is enzymatically decarboxylated to tryptamine, which serves as a biosynthetic precursor for these substances. To ensure a high metabolic flux towards DMT, 5-MeOH-DMT or beta-carbolines, enhanced levels of tryptophan are pursued. This can be achieved by manipulating the endogenous tryptophan biosynthesis pathway of the microorganism at one or more different steps of tryptophan biosynthesis.
In an embodiment the microorganism is genetically modified in one or more ways. One possible modification applies to the aroF gene (aroG gene or aroH gene) (D AHP-synthase), which forms DAHP via a condensation reaction between phosphoenolpyruvate (PEP) and erythrose-4- phosphate, is mutated by deleting its residue Ilell, making it resistant to feedback inhibition (Zhao et al. 2011). One possible modification applies to trpED gene, encoding for the anthranilate (ANTA) synthase, which catalyzes a biosynthetic step towards tryptophan, is made feedback- resistant by a S40F mutation (Zhao et al. 2011). One possible modification applies to the elimination of a gene, coding for a trp repressor ( trpR ). One possible modification applies to the elimination of a gene, coding for a tryptophan degradation enzyme ( trpR ) (Zhao et al. 2011). One possible modification applies to the elimination of genes responsible for phenylalanine (pheA ) or tyrosine ( tyrA ) production, eliminating competing pathways of tryptophan biosynthesis (Zhao et al. 2011). One possible modification increases the availability of the biosynthetic precursor phosphoenolpyruvate (PEP) by inactivation of one or more pyruvate kinases enzymes (pykA , pykF ), by the enhancement of the pckA gene, or by attenuation of the CsrA regulatory protein, by expression of its negative regulatory RNA, coded by csrB (Rodriguez et al. 2014). One possible modification involves overexpressing the genes coding for transketolase (tktA) or transaldolase (talB) or the overexpression of the enzyme glucose-6-phosphate dehydrogenase (Rodriguez et al. 2014). One possible modification attempts to reduce acetate formation by replacing the pta gene with a different variant (Liu et al. 2016). The genetically modified microorganism contains one or more genes or gene clusters, which enable the expression of enzymes used for the production of DMT, 5-MeOH-DMT, beta- carbolines from tryptophan and biosynthetic precursors such as tryptamine.
The gene or gene clusters contain one or more different combinations of the following enzymes: a. Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC), is involved in decarboxylating tryptophan to tryptamin (EC Nr.: 4.1.1.28). One possible implementation contains the enzyme isoform from Carathanthus roseus (Park et al. 2010), which is codon optimized for the respective microorganism (identified as SEQ ID NO 1):
ATGGGCAGCATTGATAGCACCAACGTGGCGATGAGCAACAGCCCGGTGGGCGAATT
TAAACCGCTGGAAGCGGAAGAATTTCGCAAACAGGCGCATCGCATGGTGGATTTTAT
TGCGGATTATTATAAAAACGTGGAAACCTATCCGGTGCTGAGCGAAGTGGAACCGG
GCTATCTGCGCAAACGCATTCCGGAAACCGCGCCGTATCTGCCGGAACCGCTGGATG
AT ATT AT GAA AGAT ATT C AGAA AGAT ATT ATT CCGGGC AT GACC AACT GGAT GAGCC
CGAACTTTTATGCGTTTTTTCCGGCGACCGTGAGCAGCGCGGCGTTTCTGGGCGAAAT
GCTGAGCACCGCGCTGAACAGCGTGGGCTTTACCTGGGTGAGCAGCCCGGCGGCGA
CCGAACTGGAAATGATTGTGATGGATTGGCTGGCGCAGATTCTGAAACTGCCGAAAA
GTTTCATGTTTAGCGGCACCGGCGGCGGCGTGATTCAGAACACCACCAGCGAAAGCA
TTCTGTGCACCATTATTGCGGCGCGCGAACGCGCGCTGGAAAAACTGGGCCCGGATA
GC ATT GGC AAACT GGT GT GCT AT GGC AGCGATC AGACCC AT ACC AT GTTTCCGAAA A
CCTGCAAACTGGCGGGCATTTATCCGAACAACATTCGCCTGATTCCGACCACCGTGG
AAACCGATTTTGGCATTAGCCCGCAGGTGCTGCGCAAAATGGTGGAAGATGATGTGG
CGGCGGGCTATGTGCCGCTGTTTCTGTGCGCGACCCTGGGCACCACCAGCACCACCG
CGACCGATCCGGTGGATAGCCTGAGCGAAATTGCGAACGAATTTGGCATTTGGATTC
ATGTGGATGCGGCGTATGCGGGCAGCGCGTGCATTTGCCCGGAATTTCGCCATTATC
TGGATGGCATTGAACGCGTGGATAGCCTGAGCCTGAGCCCGCATAAATGGCTGCTGG
CGTATCTGGATTGCACCTGCCTGTGGGTGAAACAGCCGCATCTGCTGCTGCGCGCGC
T GACC ACC AACCCGGAAT AT CT GAAGAAC AAAC AGAGCGAT CT GGAT AAAGTGGT G
GATTTTAAAAACTGGCAGATTGCGACCGGCCGCAAATTTCGCAGCCTGAAACTGTGG
CTGATTCTGCGCAGCTATGGCGTGGTGAACCTTCAGAGCCATATTCGCAGCGATGTG
GCGATGGGCAAAATGTTTGAAGAATGGGTGCGCAGCGATAGCCGCTTTGAAATTGTG GTGCCGCGCAACTTTAGCCTGGTGTGCTTTCGCCTGAAACCGGATGTGAGCAGCCTG CATGTGGAAGAAGTGAACAAGAAACTGCTGGATATGCTGAACAGCACCGGCCGCGT GTATATGACCCATACCATTGTGGGCGGCATTTATATGCTGCGCCTGGCGGTGGGCAG CAGCCTGACCGAAGAACATCATGTGCGCCGCGTGTGGGATCTGATTCAGAAACTGAC CGAT GATCTGCTGAAAGAAGCGTAA
One possible amino acid sequence of the Aromatic Amino acid decarboxylase enzyme (EC Nr. : 4.1.1.28 is identified as SEQ ID_NO 2:
MGSID S TNV AMSN SP V GEFKPLE AEEFRKQ AHRMVDFI AD YYKNVET YPV SEVEPGYL
RKRIPETAPYFPEPFDDIMKDIQKDIIPGMTNWMSPNFYAFFPATVSSAAFFGEMFSTAFN
S V GFTWV S SPAATEFEMIVMDWFAQIFKFPKSFMF SGTGGGVIQNTTSESIFCTIIA ARER
AFEKFGPDSIGKFVCYGSDQTHTMFPKTCKFAGIYPNNIRFIPTTVETDFGISPQVFRKMV
EDDVAAGYVPFFFCATFGTTSTTATDPVDSFSEIANEFGIWIHVDAAYAGSACICPEFRH
YFDGIERVDSFSFSPHKWFFAYFDCTCFWVKQPHFFFRAFTTNPEYFKNKQSDFDKW
DFKNWQIATGRKFRSLKLWLILRSYGVVNLQSHIRSDVAMGKMFEEWVRSDSRFEIVVP
RNFSFVCFRFKPDVSSFHVEEVNKKFFDMFNSTGRVYMTHTIVGGIYMFRFAVGSSFTE
EHHVRRVWDLIQKLTDDLLKEA b. Indolethylamine N-methyltransferase (INMT) is involved in methylating tryptamin (EC Nr.: 2.1.1.49 (formerly EC Nr.: 2.1.1.81)). One possible DNA sequence codes for the human enzyme isoform (Torres et al. 2019), which is codon optimized for the respective microorganism (identified as SEQ ID NO 3): ATGAAAGGCGGCTTTACCGGCGGCGATGAATATCAGAAACATTTTCTGCCGCGCGAT TATCTGGCGACCTATTATAGCTTTGATGGCAGCCCGAGCCCGGAAGCGGAAATGCTG AAATTTAACCTGGAATGCCTGCATAAAACCTTTGGCCCTGGCGGCCTTCAGGGCGAT ACCCTGATTGATATTGGCAGCGGCCCGACCATTTATCAGGTGCTGGCGGCGTGCGAT AGCTTTCAGGATATTACCCTGAGCGATTTTACCGATCGCAACCGCGAAGAACTGGAA AAATGGCTGAAGAAAGAACCGGGCGCGTATGATTGGACCCCGGCGGTGAAATTTGC GTGCGAACTGGAAGGCAACAGCGGTCGCTGGGAAGAAAAAGAAGAAAAACTGCGC GCGGCGGTGAAACGCGTGCTGAAATGCGATGTGCATCTGGGCAACCCGCTGGCGCCT GCGGT GCTGCCGCT GGCGGATTGCGT GCTGACCCTGCTGGCGATGGA AT GCGCGTGC TGTAGCCTGGATGCGTATCGCGCGGCGCTGTGCAACCTGGCGAGCCTGCTGAAACCG
GGCGGCCATCTGGTGACCACCGTGACCCTGCGCCTGCCGAGCTATATGGTGGGCAAA CGCGAATTTAGCTGCGTGGCGCTGGAAAAAGAAGAAGTGGAACAGGCGGTGCTGGA T GCGGGCTTTGAT ATTGAAC AGCT GCT GC ATAGCCCGC AGAGCTAT AGCGTGACCAA CGCGGCGAACAACGGCGTGTGCTTTATTGTGGCGCGCAAGAAACCGGGCCCGTAA The DNA sequence identified as SEQ ID NO 4 codes for one possible isoform of Indolethylamine N-methyltransferase (INMT) enzyme (EC Nr.: 2.1.1.49 (formerly EC Nr.: 2.1.1.81)):
MKGGFT GGDEY QKHFLPRD YLATYY SFDGSPSPE AEMLKFNLECLHKTF GPGGLQGDTL IDIGSGPTIYQVLAACDSFQDITLSDFTDRNREELEKWLKKEPGAYDWTPAVKFACELEG NSGRWEEKEEKLRAAVKRVLKCDVHLGNPLAPAVLPLADCVLTLLAMECACCSLDAYR AALCNLASLLKPGGHLVTTVTLRLPSYMVGKREFSCVALEKEEVEQAVLDAGFDIEQLL HSPQSYSVTNAANNGV CFIVARKKPGP
The Indolethylamine N-methyltransferase (INMT) enzyme primary sequence can contain one or more mutations in order to optimize the functionality of the protein. The mutations can include one or more of the following mutations: 254C, D28N, H46P, M206V or N245S (Torres et al. 2019)
Further possible genes or gene cluster combinations can involve one or more genes coding for one or more different isoforms of the enzymes Tryptophan hydroxy lyase (EC Nr.: 1.14.16.4), Tryptamine-5-hydroxylyase (EC Nr.: 1.14.14.1) or Hydroxyindole-O-Methyltransferase (EC-Nr. 2.1.1.4). These genes enable the production of 5-MeOH-DMT.
Another possible gene or gene cluster combination involves the introduction of enzymes, which catalyze a Pictet-Sprengler-reaction in order to form 5-Me-OH-DMT from the biosynthetic precursor tryptamine.
In various embodiments, a recombinant host cell incorporates modifications that increase the intake of precursor, tryptamine, to enable high-titer production of DMT. Preferably sufficient concentrations of tryptamine are present in the culture medium of he microorganism
In these or other embodiments, the host cell is modified for enhanced DMT production. In some embodiments, a recombinant Escherichia coli cell overexpresses one or more enzymes of the DMT pathway. In these or other embodiments, the host cell is modified for enhanced DMT production. In some embodiments, a recombinant Zymomonas mobilis cell overexpresses one or more enzymes of the DMT pathway.
In an embodiment of the microorganism, the production of DMT is improved by the addition of tryptophan to the media. Tryptophan can be fed at a final concentration of 1 g/1 or at a final concentration of 2 g/1 or at a final concentration of 3 g/1 or at a final concentration of 4 g/1 or at a final concentration of 5 g/1 or at a final concentration of 6 g/1. Tryptophan can be added at the time of inoculation or at 1 h after inoculation or at 2 h after inoculation or at 3 h after inoculation or at 6 h after inoculation or at 9 h after inoculation or at 12 h after inoculation or at 18 h after inoculation or at 24 h after inoculation or at 48 h after inoculation or at 72 h after inoculation.
In some embodiments, the culturing is carried out at 16-45°C, e.g., room temperature, 37°C, 30- 42 °C, 30-40°C, or 32-38°C.
Further provided are methods for producing DMT or DMT derivatives such as 5-Me-OH-DMT in a cell, e.g. the non-naturally occurring microorganism of the invention. In some embodiments, the method comprises recombinantly expressing at least one other member of the DMT pathway in the cell preferably Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N-methyltransferase and culturing the cell in the presence of DMT precursor molecules such as L-Tryptophan, Tryptamine and N-Methyltryptamine (NMT), thereby producing DMT or DMT derivatives in the cell.
In a preferred embodiment, methods for producing 5-Me-OH-DMT comprises recombinantly expressing at least one other member of the DMT pathway in the cell, and culturing the cell in the presence of DMT precursor molecules such as 5MeO before DMT is formed thereby producing 5-Me-OH-DMT in the cell.
The invention also concerns a method of converting a precursor product such as such as L- Tryptophan and/or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT, the method comprising culturing non-naturally occurring microorganism of the invention in a suitable culture medium under conditions suitable to induce expression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N- methyltransferase, and then harvesting the cultured cells or spent medium, thereby converting the precursor product into the target metabolic product such as DMT or 5-Me-OH-DMT. In a preferred embodiment, the method of converting a precursor product such as such as L- Tryptophan and/or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises harvesting and lysing the cultured cells, thereby producing cell lysate. Additionally, the method comprises purifying the target metabolic product i.e. DMT or DMT derivatives such as 5-Me-OH-DMT from the cell lysate, thereby producing a purified target metabolic product.
In a more preferred embodiment, the method of converting a precursor product such as such as L- Tryptophan and/or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises formulating DMT or 5-Me-OH-DMT in a pharmaceutical composition. In some embodiments, the method further comprises introducing a recombinant expression cassette to the cell prior to the expressing step. In some embodiments, the method further comprises introducing a recombinant expression cassette comprising Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N- methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)), e.g., simultaneously or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the DMT pathway.
In some embodiments, the method further comprises introducing a recombinant expression cassette to the cell prior to the expressing step. In some embodiments, the method further comprises introducing a recombinant expression cassette comprising at least Tryptophan hydroxylyase (EC Nr.: 1.14.16.4), Tryptamine-5-hydroxylyase (EC Nr.: 1.14.14.1), and/or Hydroxyindole-O-Methyltransferase (EC-Nr. 2.1.1.4) e.g., simultaneously, or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the 5-MeOH-DMT pathway.
In an embodiment the genes or gene clusters, which enable the expression of enzymes used for the production of DMT or DMT derivatives, are incorporated into the genetically engineered or enhanced microorganism via a shuttle vector plasmid. The shuttle vector plasmid can contain one or more specific promoters, one or more multiple cloning sites for the insertion of single genes or gene clusters, one or more terminators, one or more resistance cassettes (chosen from AmpR, KanaR, CamR, SpecR, TetR), an E. coli Origin of replication, an Origin of replication specific for the genetically engineered microorganism strain, a T7 IPTG inducible promoter.
In an embodiment, the shuttle vectors with the incorporated gene cassettes are transformed into the genetically engineered via electroporation and subsequently plated on agar plates containing one or more of the following antibiotics ampicillin, kanamycin, chloramphenicol, tetracycline and spectinomycin. Detection of the successful transformation of gene cassettes is accomplished via PCR and sequencing. The gene transcription and enzyme translation and activity are determined by transcriptome, proteome and metabolome analysis. Figure 1 describes a the process including genetic engineering and chemical molecules involved the invention described. In an embodiment, the microorganism can be genetically engineered via the CRISPR-Cas system. This technique enables deletions, insertions or point mutations in the genomic DNA of the microorganism.
In an embodiment the gram-negative bacterial strain Zymomonas mobilis is genetically engineered microorganism via the use of the endogenous CRISPR-Cas system.
The microorganism is genetically modified via the phage Lambda-derived Red recombination system. This technique enables deletions, insertions or point mutations in the genomic DNA of the microorganism. In an embodiment, a plasmid is used comprising the bacteriophage lambda red components.
The repair template essential for the homologous recombination is designed to have homologous arms to the genomic DNA of the microorganism where the mutation is incorporated as well as containing the desired mutation. Both the plasmid and the repair template are transformed into the microorganism. In an embodiment, the Lambda-derived Red recombination plasmids are transformed by electroporation into the microorganism and subsequently spread on agar plates, supplemented with antibiotics. By colony PCR, the target sequence is amplified with the help of the corresponding primers and individual positive colonies are then identified with Agarose Gel electrophoresis and sequencing.
Homologous directed repair is used as a method for the introduction of genetic material to make the non-natural occurring bacterial. The genetic material for the homologous directed repair contains a high degree of homology to the genetic sequence of the microorganism. Through an endogenous in cell mechanism the region with a high degree of homology gets build into the genome creating a non-naturally occurring bacteria. The homologous regions are design in a way that they flank a specific sequence region. This sequence region gets incorporated into the genome of the bacteria through homologous recombination. In an embodiment, the incorporated region contains FRT-sequences from Saccharomyces cerevisiae. The FRT sequence is used as a FLP-FRT site-specific recombination system. Gen fragments flanking two FRT sequences are incorporated by the Saccharomyces cerevisiae FLT gen, coding for the FLP recombinase enzyme (Zou et al 2012).
Identification and quantification of generated tryptamines can be performed by several methods combining chromatographic separation like LC, HPLC, UHPLC or GC and mass sensitive or photo optical detection. Chromatographic methods can include liquid (MeOH, ACN, hexan, water, acetic acid and others) or gaseous mobile phases (¾, He, N2, Ar) and liquid or solid stationary phases including silica gel, polydimethylsiloxane or reversed phase materials. Detection of DMT and related compounds can be achieved by using MS or MS/MS, including Sector mass spectrometry, time-of-flight mass spectrometry, the use of quadrupole mass analyzer, three-dimensional quadrupole ion trap, cylindrical ion trap, linear quadrupole ion trap, orbitrap or fourier transform ion cyclotron resonance and the use of an andequat detector, including electron multiplier systems, faraday cups, ion-to-photon detectors, microchannel plate detectors or inductive detectors. Other detection methods like UV-absorption, fluorescence, charged aerosol detector, evaporative light scattering detector, flame ionization detector, flame photometric detector, nitrogen phosphorus detector, atomic-emission detector, refractive index detector, radio flow detector, conductivity monitor, thermal conductivity detector, electron capture detector and photoionization detectors or combination of those principles can be applied. DMT can also be detected by using chemical reactions, the use of appropriate stains like iodine vapor, iodoplatinate, marquis reagent, nihydrin, HNCb-atmosphere, NNCD-reagent, PDAB-TS, TACOT, TCBI, vanillin reagents, Van Urk reagent or xanthydrol and the use of an authentic reference substance (Barker et al. 2012; Mulga et al. 2012).
Quantification can be achieved by the use of an internal standard containing e.g. 5-Meo-DMT, dr DMT, ds-DMT, 5-Br-DMT or 4F-DMT. By the addition of a known amount of these substances prior to sample preparation it is possible to calculate the actual amount of DMT and related compounds in each sample (Barker et al. 2013).
The products of the fermentation reaction, DMT for example, may be recovered from the fermentation broth by continuously removing a portion of the broth from the fermentation bioreactor, separating microbial cells from the broth (by filtration). Then, one or more products can be recovered from the broth simultaneously or sequentially. Preferably returned to the fermentation bioreactor, the cell free permeate remains after the one or more products have been obtained. In addition, other non-essential nutrients (e.g. vitamins) may be added to the cell free permeate before it is returned to the bioreactor to replenish the medium (as it is described on US8377665B2) The extraction of tryptamines derivatives from the medium or harvested cells is performed by homogenization of the cells and medium by using mechanical, physical or chemical methods. In another embodiment, the extraction of tryptamines derivatives from the medium or harvested cells is not performed homogenization. In an embodiment, two-phase extraction method using a non-polar solvent after the basification of the aqueous phase or by a direct extraction from the material using polar or nonpolar solvents. In another embodiment, solid-phase extraction (SPE) is used for DMT enrichment in a first step. Further purification can be achieved by recrystallization or precipitation using a suitable solvent for counter ion system, distillation or sublimation of DMT as well as using typical preparative chromatographic methods. Then, DMT is stored as a salt or as the free base protected from light and oxygen under an inert gas atmosphere, in a solid form or as a solution in water, ethanol, methanol or other solvents. In an embodiment, the non-naturally occurring bacteria is grown with 15N ammonium minimal medium. Enrichment of Tryptamines with isotopes (2H, 3H, nC, 13C, 14C, 13N, 15N) can be achieved by the restriction of elemental supply to an enriched source of the corresponding element. Those isotopes modified derivatives can be used for analytical, diagnostical, medicinal or other purposes.
In some embodiments, the method further comprises introducing the recombinant expression cassette to the cell prior to the expressing step. In some embodiments, the method further comprises introducing a recombinant expression cassette comprising Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N- methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)), e.g., simultaneously or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the DMT pathway. In some embodiments, the method further comprises introducing the recombinant expression cassette to the cell prior to the expressing step. In some embodiments, the method further comprises introducing a recombinant expression cassette comprising Tryptophan hydroxylyase (EC Nr.: 1.14.16.4), Tryptamine-5 -hydroxylyase (EC Nr.: 1.14.14.1), and Hydroxyindole-O- Methyltransferase (EC-Nr. 2.1.1.4) e.g., simultaneously, or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the 5- MeOH-DMT pathway.
In some embodiments, the method further comprises harvesting the DMT emitted from the cell. In some embodiments, the method further comprises harvesting the 5-Me-OH-DMT emitted from the cell.
In some embodiments, the method results in an increase in the amount of DMT produced by the cell compared to a control cell not recombinant' expressing the at least one member of the DMT pathway. For example, the method results in at least a 2-, 5-, 6-, 8-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 200-, 500-, 600-, 1000-, 1200-, 1600-, 2000-fold or higher fold increase in the amount of DMT produced by the cell compared to a control cell not recombinantly expressing the at least one member of the DMT pathway.
In some embodiments, the method results in an increase in the amount of 5-Me-OH-DMT produced by the cell compared to a control cell not recombinant' expressing the at least one member of the 5-Me-OH-DMT pathway. For example, the method results in at least a 2-, 3-, 5-, 6-, 8-, 10, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 200-, 500-, 600-, 1000-, 1200-, 1600-, 2000- fold or higher fold increase in the amount of 5-Me-OH-DMT produced by the cell compared to a control cell not recombinantly expressing the at least one member of the 5-Me-OH-DMT pathway.
In some embodiments, the method results in a cessation of cell growth, i.e., the cells no longer replicate, or grow/ replicate at a greatly reduced rate. In some embodiments, the method results in cell growth 20, 10, 5, 1% or lower compared to a control cell not recombinantly expressing at least one member of the DMT pathway. In some embodiments the precursor molecule, tryptamine, is quantitatively converted to DMT, e.g., with an efficiency of 18, 25, 30, 50, 60, 70, 80% or higher. In some embodiments, the mass ratio of DMT to dry cell weight (Isp/DCW) is at least 0.25, 0.5, 0.7, 0.8, 0.9, 1 .0 or higher. In some embodiments, the DMT -to-biomass (w:w) ratio is at least 0.3%, 0.5%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 1 1.0%, 12.0%, 13.0%, 14.0%, 15.0% or more.
In some embodiments, the method results in a cessation of cell growth, i.e., the cells no longer replicate, or grow/ replicate at a greatly reduced rate. In some embodiments, the method results in cell growth 20, 10, 5, 1% or lower compared to a control cell not recombinantly expressing at least one member of the 5-Me-OH-DMT pathway. In some embodiments the precursor molecule, tryptamine, is quantitatively converted to 5-Me-OH-DMT, e.g., with an efficiency of 18, 25, 30, 50, 60, 70, 80% or higher. In some embodiments, the mass ratio of 5-Me-OH-DMT to dry cell weight (Isp/DCW) is at least 0.25, 0.5, 0.7, 0.8, 0.9, 1 .0 or higher. In some embodiments, the DMT -to-biomass (w:w) ratio is at least 0.3%, 0.5%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 1 1.0%, 12.0%, 13.0%, 14.0%, 15.0% or more. In some aspects, the present invention provides methods for making a product comprising DMT, 5-Me-OH-DMT or DMT and 5-Me-OH-DMT. In various aspects, the product is a pharmaceutical composition, a dietary supplement, beverages or a baked good. The DMT and 5-Me-OH-DMT of the present invention can be mixed with one or more excipients to form a pharmaceutical product, which may be a pill, a capsule, a mouth spray, or an oral solution.
In an embodiment, shuttle vectors with the incorporated genes (Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) and Indolethylamine N- methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81)) or Tryptophan hydroxylyase (EC Nr.: 1.14.16.4), Tryptamine-5 -hydroxylyase (EC Nr.: 1.14.14.1)) are transformed into Zymomonas mobilis via electroporation and subsequently plated on agar plates containing one or more of the following antibiotics ampicillin, kanamycin, chloramphenicol, tetracycline and spectinomycin. Detection of the successful transformation of gene cassettes is accomplished via PCR and sequencing. The gene transcription and enzyme translation and activity are determined by transcriptome, proteome and metabolome analysis.
The preferred embodiment of the invention are listed below in a claim format
1. A non-naturally occurring microorganism modified to produce N,N-Dimethyltryptamine. 2. A non-naturally occurring microorganism according to embodiment 1 expressing the enzymes: a. Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) b. Indolethylamine N-methyltransferase (INMT) (EC Nr. : 2.1.1.49)'
3. A non-naturally occurring microorganism according to embodiment 1 or 2 expressing a. the Aromatic Amino acid decarboxylase enzyme is a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 2 and wherein the Aromatic Amino acid decarboxylase enzyme retains Tryptamine 4-monooxygenase activity, and b. the Indolethylamine N-methyltransferase (INMT) enzyme is a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 4 and wherein the Indolethylamine N-methyltransferase retains Isoform of Indolethylamine N-methyltransferase (INMT) enzyme activity.
4. A non-naturally occurring microorganism according to any of embodiments 1 to 3 comprising a. a nucleic acid sequence encoding Tryptophan Decarboxylase where in the sequence of the nucleic acid encoding Tryptophan Decarboxylase is at least 80% identical to nucleic acid sequence of SEQ ID No. 1. b. a nucleic acid sequence encoding Indolethylamine N-methyltransferase where in the sequence of the nucleic acid encoding Indolethylamine N-methyltransferase is at least 80% identical to the nucleic acid sequence of SEQ ID No. 3.
5. A non-naturally occurring microorganism according to embodiment 4 comprising an inducible promoter operably linked to the nucleic acid encoding Tryptophan Decarboxylase and/or to the nucleic acid encoding Indolethylamine N-methyltransferase.
6. A non-naturally occurring microorganism according any of embodiments 1-5 modified to produce 5-MeOH- N,N-Dimethyltryptamine 7. A non-naturally occurring microorganism according to any of embodiments 1-6 wherein the microorganism is optimized for a higher production of Tryptophan.
8. A non-naturally occurring microorganism according to any of embodiments 1-7 wherein the microorganism is able to grow in ammonium minimal medium.
9. A non-naturally occurring microorganism according to any of the preceding embodiments wherein the microorganism is a bacterium or a yeast.
10. A non-naturally occurring microorganism according to embodiment 9 wherein the bacterium is selected from the genus Escherichia, Sccharomyces, Clostridium, Bacillus,
Lactococcus, Zymomonas, Corynebacterium, Pichia, Candida, Hansenula, Trichoderma, Acetobacterium, Ralstonia, Cupravidor, Salmonella, Klebsiella, Paenibacillus, Lactobacillus, Rhodococcus, Enterococcus, Alkaligenes, Brevibacterium, Methylobacterium, Methylobacterium, Methylococcus, Methylomonas, Methylocystis and Methylosinus. 11. A non-naturally occurring microorganism according to embodiment 10 wherein the bacterium is selected from the genus Escherichia and Zymomonas.
12. A non-naturally occurring microorganism according to embodiment 11 wherein the bacterium is Escherichia coli or Zymomonas mobilis.
13. Method of converting L-Tryptophan and/or Tryptamine into DMT and/or 5-Me-OH- DMT, the method comprising culturing non-naturally occurring microorganism of any of embodiments 1-12 in a suitable culture medium under conditions suitable to induce expression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N-methyltransferase, and then harvesting the cultured cells or spent medium, thereby converting -Tryptophan and/or Tryptamine into DMT and/or 5- Me-OH-DMT.
14. Method of embodiment 13 further comprising the step of : a) lysing the non-naturally occurring microorganism b) purifying DMT and/or 5-Me-OH-DMT from the cell lysate, thereby producing a purified target metabolic product.
15. Method of embodiment 14 further comprising formulating DMT or 5-Me-OH-DMT in a pharmaceutical composition.
DETAILED DESCRIPTION OF THE INVENTION Introduction
DMT and 5-Me-OH-DMT, compounds of interest, are produced using an expression system as described herein that employs, as well as analogs of such compounds. In some embodiments, each step of a metabolic pathway that produces the DMT and 5-Me-OH-DMT, compound of interests, occurs in a modified recombinant cell described herein. In other embodiments, at least one step of the metabolic pathway occurs in a modified recombinant cell described herein, and at least one step of the metabolic pathway occurs extracellularly, e.g., in microorganism media or within a co-cultured modified recombinant cell. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. Thus, for example, some embodiments may encompass a host cell "comprising" a number of components, other embodiments would encompass a host cell "consisting essentially of the same components, and still other embodiments would encompass a host cell "consisting of the same components. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The nucleic acid sequences provided by the invention is in the form of an expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding polypeptide being involved in DMT and 5-Me-OH-DMT synthesis.
The polynucleotides provided by the invention can either be isolated from their natural genomic environment, modified after their isolation, or produced artificially from pure sequence information.
Definitions
Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art to which the present application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
As used herein and in the appended claims, the singular forms“a,”“and,” and „the” include plural referents unless the context clearly dictates otherwise.
The term "genome" or "genomic DNA" is referring to the heritable genetic information of a host organism. Said genomic DNA comprises the entire genetic material of a cell or an organism. In the case of eukaryotic organisms, the terms genome or genomic DNA refers to the total amount of DNA of a cell, including the DNA of the nucleus (chromosomal DNA), extrachromosomal DNA, and organellar DNA (e.g. of mitochondria). Preferably, the terms genome or genomic DNA, when used in context of eukaryotic organisms, is referring to the chromosomal DNA of the nucleus or genomic DNA or heterologous plasmid DNA. The terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to a single or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The monomer is typically referred to as a nucleotide. Nucleic acids can include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.
As used herein, the terms „abouf ’ and „around” indicate a close range around a numerical value when used to modify that specific value. If ‘X” were the value, for example, “about X” or „around X” would indicate a value from 0.9X to 1.1X, e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or a value from 0.99X to 1.01X. Any reference to “about X” or „around X” specifically indicates at least the values X, 0.9X, 0.9 IX, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.IX, and values within this range.
The phrase "nucleic acid sequence encoding" or a "nucleic acid coding sequence" refers to a nucleic acid which directs the expression of a specific protein or peptide. Such nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA, and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. In some embodiments, the nucleotide sequence is codon-optimized to reflect the typical codon usage of the host cell without altering the polypeptide encoded by the nucleotide sequence. In certain embodiments, the term "codon optimization" or "codon-optimized" refers to modifying the codon content of a nucleic acid sequence without modifying the sequence of the polypeptide encoded by the nucleic acid to enhance expression in a particular host cell. In certain embodiments, the term is meant to encompass modifying the codon content of a nucleic acid sequence as a means to control the level of expression of a polypeptide ( e.g. ., either increase or decrease the level of expression). Accordingly, described are nucleic sequences encoding the enzymes involved in the engineered metabolic pathways. In some embodiments, a non-naturally occurring microorganism may express one or more polypeptide having an enzymatic activity necessary to perform the steps described below.
For example, a particular cell may comprises one, two, three, four, five or more than five nucleic acid sequences, each one encoding the polypeptide(s) necessary to produce DMT or 5-Me-OH- DMT compound, or compound intermediate described herein.
Alternatively, a single nucleic acid molecule can encode one, or more than one, polypeptide. For example, a single nucleic acid molecule can contain nucleic acid sequences that encode two, three, four or even five different polypeptides.
Nucleic acid sequences useful for the invention described herein may be obtained from a variety of sources such as, for example, amplification of cDNA sequences, DNA libraries, de novo synthesis, excision of genomic segment. The sequences obtained from such sources may then be modified using standard molecular biology and/or recombinant DNA technology to produce nucleic sequences having desired modifications. Exemplary methods for modification of nucleic acid sequences include, for example, site directed mutagenesis, PCR mutagenesis, deletion, insertion, substitution, swapping portions of the sequence using restriction enzymes, optionally in combination with ligation, homologous recombination, site specific recombination or various combination thereof. In other embodiments, the nucleic acid sequences may be a synthetic nucleic acid sequence. Synthetic polynucleotide sequences may be produced using a variety of methods described in U.S. Patent No. 7,323,320, as well as U.S. Pat. Appl. Pub. Nos. 2006/0160138 and 2007/0269870.
The term "promoter" refers to a polynucleotide which directs the transcription of a structural gene to produce mRNA. Typically, a promoter is located in the 5' region of a gene, proximal to the start codon of the coding region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent or by the induced release of a suppressor. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter. A polynucleotide is "heterologous to" an organism or a second polynucleotide if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e. g. a genetically engineered coding sequence or an allele from a different ecotype, variety or strain).
"Transgene", "transgenic" or "recombinant" refers to a polynucleotide manipulated by man or a copy or complement of a polynucleotide manipulated by man. For instance, a trans- genie expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of manipulation by man (e.g., by methods described in Sambrook et al., Molecular Cloning- A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, restriction sites or plasmid vector sequences manipulated by man may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.
Typically, the plasmid contains, one or more resistance cassettes (selected from ampicillin resistance (AmpR), kanamycin resistance (KanaR), chloramphenicol resistance (CamR), spectinomycin resistance (SpecR), tetracycline resistance (TetR)) and an Zymomonas mobilis Origin of Replication (OR) or Escherichia coli Origin or Replication (OR).
In case the term "recombinant" is used to specify an organism or cell, e.g. a microorganism, it is used to express that the organism or cell comprises at least one "transgene", "transgenic" or "recombinant" polynucleotide, which is usually specified later on. The terms "operable linkage" or "operably linked" are generally understood as meaning an arrangement in which a genetic control sequence, e.g. a promoter, enhancer or terminator, is capable of exerting its function with regard to a polynucleotide being operably linked to it, for example a polynucleotide encoding a polypeptide. Function, in this context, may mean for example control of the expression, i.e. transcription and/or translation, of the nucleic acid sequence. Control, in this context, encompasses for example initiating, increasing, governing, or suppressing the expression, i.e. transcription and, if appropriate, translation. Controlling, in turn, may be, for example, tissue and / or time specific. It may also be inducible, for example by certain chemicals, stress, pathogens and similar.
Preferably, operable linkage is understood as meaning for example the sequential arrangement of a promoter, of the nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator, in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed. An operably linkage does not necessarily require a direct linkage in the chemical sense. Genetic control sequences such as, for example, enhancer sequences are also capable of exerting their function on the target sequence from positions located at a distance to the polynucleotide, which is operably linked. Preferred arrangements are those in which the nucleic acid sequence to be ex- pressed is positioned after a sequence acting as promoter so that the two sequences are linked covalently to one another. The distance between the promoter and the amino acid sequence encoding polynucleotide in an expression cassette, is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. The skilled worker is familiar with a variety of ways in order to obtain such an expression cassette. However, an expression cassette may also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for example an endogenous promoter, for example by means of homologous recombination or else by random insertion. Such constructs are likewise understood as being expression cassettes for the purposes of the invention.
The term "flanking regions" refers to regions or sequences located upstream and/or downstream of a nucleic acid coding sequence in a recombinant expression cassette which is involved in double homologous recombination (e.g., integration) of a portion of the cassette with a host cell's genome. The term "double homologous recombination" refers to the ability of nucleic acid sequences to exchange, wherein a nucleic acid stably integrates into the genome of a host cell's DNA sequence to make a new combination of DNA sequence.
The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity can be partial, in which only some of the nucleic acids match according to base pairing, or complete, where ail the nucleic acids match according to base pairing.
The terms "protein", "peptide", and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid metics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutaniate, and O- phosphoserme. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
A “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and/or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively- charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic amino acids Gly, Ala, Val, Leu and He; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro; (viii) small hydroxyl amino acids Ser and Thr; and sulfur-containing amino acids Cys and Met. Reference to the charge of an amino acid in this paragraph refers to the charge at pH 7 0
"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Conservatively modified variants can include polymorphic variants, interspecies homologs (orthologs), intraspecies homologs (paralogs), and allelic variants.
The term "% identity" and its derivatives are used interchangeably herein with the term "% homology" and its derivatives to refer to the level of a nucleic acid or an amino acid sequence’s identity between another nucleic acid sequence or any other polypeptides, or the polypeptide's amino acid sequence, where the sequences are aligned using a sequence alignment program. In the case of a nucleic acid the term also applies to the intronic and/or intergenic regions. The terms "identical" or % "identity," in the context of two or more nucleic acids or proteins, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g. the NCBI web site at ncbi.nlm.nih.gov/BLAST/. Such sequences are then said to be "substantially identical." This definition also refers to, and can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Optimal alignment of such sequences can be carried out by any of the publically available algorithms or programs for determining sequence identity and alignment, e.g., BLAST.
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al, BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motifts), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).
The term "cassette" or "expression cassette" means those constructs in which the nucleic acid sequence encoding an amino acid sequence to be expressed is linked operably to at least one genetic control element which enables or regulates its expression (i.e. transcription and / or translation). An expression cassette typically includes a sequence to be expressed, and sequences necessary for expression of the sequence to be expressed. The sequence to be expressed can be a coding sequence or a non-coding sequence {e.g., an inhibitory sequence). Generally, an expression cassette is inserted into an expression vector (e.g., a plasmid) to be introduced into a host cell. The expression may be, for example, stable or transient, constitutive, or inducible. Expression cassettes may also comprise the coding regions for two or more polypeptides and lead to the transcription of polycistronic RNAs. The term "effectively binds to ribosomes" or "effectively recruits ribosomes," in reference to an RBS, indicates that the RBS binds to ribosomes in the relevant cell or expression system in a manner sufficient to initiate translation. For example, an RBS in a bacterial (e.g., E. cob) cell is selected to bind to bacterial (Escherichia cob) ribosomes (e.g., the 16S rRNA), an RBS in a cyanobacterial cell (e.g., Synechocystis) is selected to bind to cyanobacterial ribosomes (e.g., the 16S rRNA) etc. One of skill will appreciate that the cell or expression system can be manipulated to include heterologous ribosomes that bind to a particular RBS.
The terms "transfection" and "transformation" refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 18.1- 18.88.
A polynucleotide or polypeptide sequence is "heterologous to" an organism or a second sequence if it originates from a different species, or, if from the same species, it is modified from its original form. For example, a promoter operability linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety). Similarly, a heterologous expression cassette includes sequence!) that are from a different species than the cell into which the expression cassette is introduced, or if from the same species, is genetically modified. Yeast or other eukaryotic species may be introduced on high- level expression plasmid vectors or through genomic integration using methods well known to those skilled in the art. Such methods may involve CRISPR Cas-9 technology, yeast artificial chromosomes (YACs) or the use of retrotransposons. Alternatively, if natural to the host organism, such genes may be up regulated by genetic element integration methods known to those skilled in the art.
"Recombinant" refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated using well known methods. A recombinant expression cassette comprising a promoter operability linked to a second polynucleotide (e.g., a coding sequence) can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994- 1998)). A recombinant expression cassette (or expression vector) typically comprises polynucleotides in combinations that are not found in nature. For instance, human manipulated restriction sites or plasmid vector sequences can flank or separate the promoter from other sequences. A recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).
The terms "culture," "culturing," "grow," "growing," "maintain," "maintaining," "expand," "expanding," etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside its normal environment under controlled conditions, e.g., under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, stasis, differentiation or division. The term does not imply that all cells in the culture survive, grow, or divide, as some may naturally die or senesce. Cells are typically cultured in media, which can be changed during the course of the culture. The terms "media" and "culture solution" refer to the cell culture milieu. Media is typically an isotonic solution, and can be liquid, gelatinous, or semi-solid, e.g., to provide a matrix for cell adhesion or support. Media, as used herein, can include the components for nutritional, chemical, and structural support necessary for culturing a cell. Typically, media includes a carbon source for biosynthesis and metabolism. In the case of plant or other photosynthetic cell cultures, the carbon source is typically CO2.
A "control," e.g., a control cell, control sample, or control value, refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample or condition. For example, a test sample can include cells exposed to a test condition or a test agent, while the control is not exposed to the test condition or agent (e.g., negative control). The control can also be a positive control, e.g., a known cell exposed to known conditions or agents, for the sake of comparison to the test condition. For example, a positive control can include a cell with a known level of production of the product of interest. A control can also represent an average value gathered from a plurality of samples, e.g., to obtain an average value. A control value can also be obtained from the same cell or population of cells, e.g., from an earlier-obtained sample, prior to the disorder or deficiency, or prior to treatment. One of skill will recognize that controls can be designed for assessment of any number of parameters. One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data.
A tryptamine is a monoamine alkaloid, which contains an indole ring structure. It is structurally similar to the amino acid tryptophan from which the name derives. Tryptamine is found in trace amounts in the brains of mammals and is hypothesized to play a role as a neuromodulator or neurotransmitter. Similar to other trace amines tryptamine binds to human trace amine-associated receptor 1 (TAARl) as an agonist. TAAR1 plays a significant role in regulating neurotransmission in dopamine norepinephrine and serotonin neurons in the CNS. Additionally, it also affects immune system and neuroimmune system function through different mechanisms. (Jones RS. Tryptamine: a neuromodulator or neurotransmitter in mammalian brain Ί . Prog Neurobiol. 1982;19(1-2):117-139. doi: 10.1016/0301 -0082(82)90023-5) (Khan MZ, Nawaz W. The emerging roles of human trace amines and human trace amine-associated receptors (hTAARs) in central nervous system . Biomed Pharmacother. 2016;83:439-449. doi:10.1016/j.biopha.2016.07.002) (Rogers TJ. The molecular basis for neuroimmune receptor signaling. J Neuroimmune Pharmacol. 2012;7(4):722-724. doi:10.1007/sl 1481-012-9398-4).
RECOMBINANT METHODS FOR PRODUCTION OF DMT AND 5-Me-OH-DMT Methods of transformation of microorganisms such as bacteria cells are well known in the art.
Culture conditions such as expression time, temperature, and pH can be controlled so as to afford target DMT and 5-Me-OH-DMT in high yield. Host cells are generally, but not necessarily, cultured in the presence of starting materials, such as tryptamines, hexanoic acid, prenol, isoprenol, or the like, for periods of time ranging from a few hours to a day or longer (e.g., 24 hours, 30 hours, 36 hours, or 48 hours) at temperatures ranging from about 20 °C to about 40 °C depending on the particular host cells employed. As explained above, in some embodiments, host cells, transformed or genomically integrated with plasmids or vectors containing at least one or more than one expression cassette.
The cells used to produce DMT or 5-Me-OH-DMT as described herein are genetically modified. That is, heterologous nucleic acid is introduced into the cells. The genetically modified cells do not occur in nature. Suitable cells are capable of expressing a nucleic acid construct (expression cassette) encoding biosynthetic enzymes, as described herein. In some embodiments, the cell naturally produces at least some biosynthetic precursors, e.g., tryptamine. In some embodiments, e.g., those involving DMT or 5-Me-OH-DMT production via the DMT or 5-Me-OH-DMT pathway, genes encoding desired enzymes can be heterologous to the cell, or native to the cell but operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the cell.
Any microorganism can be used in the present method so long as it remains viable after being transformed with the heterologous genes.
Microorganisms used for producing the DMT or 5-Me-OH-DMT, e.g., microorganisms lacking the DMT and 5-Me-OH-DMT pathway, e.g., bacteria, cyanobacteria or green microalgae, are engineered to express heterologous enzymes that generate DMT and 5-Me-OH-DMT.
The nucleic acid constructs described herein can be operably linked to a promoter and/or terminator so that the desired transcript(s) and protein(s) are expressed in a cell cultured under suitable conditions. Methods for designing and making nucleic acid constructs and expression vectors are well known to those skilled in the art.
Sequences of nucleic acids encoding the subject enzymes are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning. For example, in direct chemical synthesis, oligonucleotides of up to about 40 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence. Further, commercial services are available that can supply synthetic genes of the desired sequence. In addition, the desired sequences may be isolated from natural sources using well known cloning methodology, e.g., employing PGR to amplify the desired sequences and join the amplified regions.
The nucleic acid coding sequences for desired biosynthetic enzymes can be incorporated into an expression cassette. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a nucleic acid sequence into an expression cassette, and into an expression vector for introduction to a cell. A typical expression vector contains the desired nucleic acid sequence preceded by one or more regulatory regions (e.g., promoter), along with a ribosome binding site (RBS). Promoters can be either constitutive or inducible, e.g., under certain environmental conditions.
As will be appreciated by those of ordinary skill in the art, the invention is not limited with respect to the precise promoter or expression vector used. Although any suitable expression vector may be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pET, pGex, pJF119EH, pSClOl, pBR322, pBBRlMCS-3, pUR, EX, pMRIOO, pCR4, pBAD24, pUC 19; and bacteriophages, such as Ml 3 phage and l phage. Certain expression vectors may only be suitable for particular host cells which can be readily determined by one of ordinary skill in the art. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the literature, which describe expression vectors and their suitability to any particular host cell.
Homologous recombination can occur between, the expression vector and the homologous region in one or more genomic copies present in the host cell. Typically, a selectable marker present on the expression vector is used to isolate transformant cells having undergone double homologous recombination by a selection method, such as antibiotic resistance or drug resistance.
Typically, the plasmid contains one or more specific promoters, one or more multiple cloning sites for the insertion of single genes or gene clusters, one or more terminators, one or more resistance cassettes (selected from ampicillin resistance (AmpR), kanamycin resistance (KanaR), chloramphenicol resistance (CamR), spectinomycin resistance (SpecR), tetracycline resistance (TetR)) and an Zymomonas mobilis Origin of Replication (OR) and Escherichia coli Origin of Replication. Detection of the successful transformation of gene cassettes can be accomplished via PCR and sequencing. The gene transcription and enzyme translation and activity are determined by transcriptome, proteome and metabolome analysis. Cell culture techniques are commonly known in the art and described, e.g., in Sambrook, et al. (1989) Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Cells are typically cultured in isotonic media that includes a carbon source, and in some cases, selection factors to select for recombinant cells (e.g., those with antibiotic resistance).
The host cell is preferably cultured at a temperature between 22° C and 37° C. While commercial biosynthesis in host cells such as E. coli can be limited by the temperature at which overexpressed and/or foreign enzymes (e.g., enzymes derived from plants) are stable, recombinant enzymes (including the terpenoid synthase) may be engineered to allow for cultures to be maintained at higher temperatures, resulting in higher yields and higher overall productivity. In some embodiments, the host cell (bacterial or yeast host cell) is cultured at about 22° C or greater, about 23° C or greater, about 24° C or greater, about 25° C or greater, about 26° C or greater, about 27° C or greater, about 28° C or greater, about 29° C or greater, about 30° C or greater, about 31° C or greater, about 32° C or greater, about 33° C or greater, about 34° C or greater, about 35° C or greater, about 36° C or greater, or about 37° C.
DMT and 5-Me-OH-DMT can be extracted from media and/or whole cells and recovered. The DMT or 5-Me-OH-DMT are recovered and optionally purified by fractionation (e.g. fractional distillation, chromatography, etc....). The product can be recovered by any suitable process, including partitioning the desired product into an organic phase. The production of the desired product can be determined and/or quantified, for example, by gas chromatography (e.g., GC) or high-pressure liquid chromatography (HPLC-)-.in combination with a suitable detection method, preferably mass spectrometry (MS) or other systems (FID, RI, DAD) The desired product can be produced in batch or continuous bioreactor systems.
The amounts of DMT and 5-Me-OH-DMT can be measured in a recombinant host cell to identify rate limiting steps in the biosynthetic pathway. Once a rate-limiting step has been identified, expression or activity of the (limiting) one or more enzyme can be modified by various methods known in the art, such as codon optimization, use of a stronger or weaker promotor, expressing multiple copies of the corresponding gene, and constructing variants with increase stability and/or activity or to knock-out existing genes that might prevent high yield or production.
Identification and quantification of DMT and 5-Me-OH-DMT can be performed by several methods combining chromatographic separation; for example LC, HPLC, UHPLC or GC, and mass sensitive or photo optical detection. Chromatographic methods can include liquid (MeOH, ACN, hexan, water, acetic acid and others) or gaseous mobile phases (¾, He, N2, Ar) and liquid or solid stationary phases including silica gel, polydimethylsiloxane or reversed phase materials. Detection of DMT and related compounds can be achieved by using MS or MS/MS including sector mass spectrometry, time-of-flight mass spectrometry, the use of quadrupole mass analyzer, three-dimensional quadrupole ion trap, cylindrical ion trap, linear quadrupole ion trap, orbitrap or fourier transform ion cyclotron resonance and the use of an andequat detector, including electron multiplier systems, faraday cups, ion-to-photon detectors, microchannel plate detectors or inductive detectors. Other detection methods like UV-absorption, fluorescence, charged aerosol detector, evaporative light scattering detector, flame ionization detector, flame photometric detector, nitrogen phosphorus detector, atomic-emission detector, refractive index detector, radio flow detector, conductivity monitor, thermal conductivity detector, electron capture detector and photoionization detectors or combination of those principles can be applied. DMT and 5-Me-
OH-DMT can also be detected by using chemical reactions, the use of appropriate stains like iodine vapor, iodoplatinate, marquis reagent, nihydrin, HN03-atmosphere, NNCD-reagent, PDAB-TS, TACOT, TCBI, vanillin reagents, Van Urk reagent or xanthydrol and the use of an authentic reference substance (Barker et al. 2012 DOI 10.1002/dta.422) (Mulga et al. 2007 ISBN 0-9770876-5-4).
Quantification can be achieved by the use of an internal or external standard containing DMT and 5-Me-OH-DMT or isotope labeled derivatives. By the addition of a known amount of these substances prior to sample preparation, it is possible to calculate the actual amount of DMT and 5-Me-OH-DMT and related compounds in each sample (Barker et al. 2013
DOI 10.1002/bmc.2981 ). In other embodiments, DMT and 5-Me-OH-DMT are produced simultaneously produced by a recombinant host cell are retained within the recombinant cell. DMT and 5-Me-OH-DMT can be recovered from the culture medium or from the recombinant host cell.
The invention also concerns a method of converting L-Tryptophan or Tryptamine into a target metabolic product such as DMT or 5-Me-OH-DMT, the method comprising culturing non- naturally occurring microorganism of the invention in a suitable culture medium under conditions suitable to induce expression of Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N-methyltransferase, and then harvesting the cultured cells or spent medium, thereby converting the precursor product into the target metabolic product. In a preferred embodiment, the method of converting a precursor product such as such as L-Tryptophan, Tryptamine and N-Methyltryptamine (NMT) into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises harvesting and lysing the cultured cells, thereby producing cell lysate. Additionally, the method comprises purifying the target metabolic product i.e. DMT or DMT derivatives such as 5-Me-OH-DMT from the cell lysate, thereby producing a purified target metabolic product.
In a more preferred embodiment, the method of converting a precursor product such as such as L- Tryptophan, Tryptamine and N-Methyltryptamine (NMT) into a target metabolic product such as DMT or 5-Me-OH-DMT further comprises formulating DMT or 5-Me-OH-DMT in a pharmaceutical composition.
The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
With the above context, the following consecutively numbered embodiments provide further specific aspects of the invention:
1. A non-naturally occurring microorganism modified to produce N,N-Dimethyltryptamine. 2. A non-naturally occurring microorganism according to embodiment 1 modified to comprise the enzymes: a. Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) b. Indolethylamine N-methyltransferase (INMT) (EC Nr.: 2.1.1.49 (EC Nr.: 2.1.1.81))
3. A non-naturally occurring microorganism according to embodiment 1 modified to produce 5-MeOH- N,N-Dimethyltryptamine
4. A non-naturally occurring microorganism according to embodiment 3 comprising one or more genes coding for one or more different isoforms of: a. Tryptophan hydroxylyase (EC Nr.: 1.14.16.4) b. Tryptamine-5-hydroxylyase (EC Nr. : 1.14.14.1), and c. Hydroxyindole-O-Methyltransferase (EC-Nr. 2.1.1.4)
5. A non-naturally occurring microorganism according to embodiment 1 wherein the microorganism is optimized for a higher production of Tryptophan.
6. A non-naturally occurring microorganism according to embodiment 1 wherein the microorganism is able to grow in ammonium minimal medium.
7. A non-naturally occurring microorganism according to embodiment 3 wherein the microorganism is able to produce N,N-Dimethyltryptamine from ammonium minimal medium.
8. A non-naturally occurring microorganism according to embodiment 8 wherein the microorganism is a bacterium.
9. A non-naturally occurring microorganism according to embodiment 5 wherein the bacterium is selected from the genus Escherichia, Sccharomyces, Clostridium, Bacillus, Lactococcus, Zymomonas, Corynebacterium, Pichia, Candida, Hansenula, Trichoderma, Acetobacterium, Ralstonia, Cupravidor, Salmonella, Klebsiella, Paenibacillus, Lactobacillus, Rhodococcus, Enterococcus, Alkaligenes, Brevibacterium, Methylobacterium, Methylobacterium, Methylococcus, Methylomonas, Methylocystis and Methylosinus.
10. A non-naturally occurring microorganism according to embodiment 9 wherein the bacterium is selected from the genus Escherichia and Zymomonas. 11. A non-naturally occurring microorganism according to embodiment 10 wherein the bacterium is Escherichia coli.
12. A non-naturally occurring microorganism according to embodiment 10 wherein the bacterium is Zymomonas mobilis.
Figure Description
Figure 1 : Presentation of the process including genetic engineering and chemical molecules involved in the invention as described. Figure 1 describes the concept of the invention: A biosynthetic system made of a modified cell which is modified with a vector to comprise the required sequences to acquire the substrates (e.g. Tryptophan) present in the medium and produce DMT.
Figure 2: FC/MS/MS chromatogram showing tryptophan (peak at 4.5 min) in samples prepared from a DMT producing, modified microorganism.
Figure 3: FC/MS/MS chromatogram showing tryptamine (peak at 5.2 min) in samples prepared from a DMT producing, modified microorganism. Figure 4: FC/MS/MS chromatogram showing N-methyltryptamine (peak at 5.9 min) in samples prepared from a DMT producing, modified microorganism.
Figure 5: FC/MS/MS chromatogram showing N,N-dimethyltryptamine (peak at 6.1 min) in samples prepared from a DMT producing, modified microorganism.
Figure 6: FC/MS/MS chromatogram showing tryptophan (peak at 4.5 min) in a sample prepared from commercial tryptophan reference material.
Figure 7: FC/MS/MS chromatogram showing tryptamine (peak at 5.2 min) in samples prepared from commercial tryptamine reference material. Figure 8: LC/MS/MS chromatogram showing N-methyltryptamine (peak at 5.9 min) in samples prepared from samples prepared from commercial NMT reference material.
Figure 9: LC/MS/MS chromatogram showing N,N-dimethyltryptamine (peak at 6.1 min) in samples prepared from commercial DMT reference material.
Figure 10: Ions of DMT used for MRM in LC7ESI/MS/MS: The protonated DMT can undergo alpha- or beta-cleavage leading to the fragments m/z= 144 and m/z=58 {Chen, B.-H.; Liu, J.-T.; Chen, H.-M.; Chen, W.-X; Lin, C.-H. Comparison of the Characteristic Mass Fragmentations of Phenethylamines and Tryptamines by Electron Ionization Gas Chromatography Mass Spectrometry, Electrospray and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. Appl. Sci. 2018, 8, 1022.).
Figure 11 : Signal for DMT when injecting increasing concentrations of DMT reference material.
Figure 12: Signal for NMT over the time of the cultivation.
Figure 13: Signal for DMT over the time of the cultivation. Figure 14: Signal for tryptophan over the time of the cultivation.
Proof of concept DMT of biosynthesis from glucose in E.coli (E. coli (BL21) + pCDFDuet TdcCro OOl InmtHsa OOl (K2)) In order to proof the formation of DMT in our modified microorganism we performed a cultivation using a minimal medium, our strain, a 3.0 litter benchtop bioreactor system and LC/MS/MS. After an initial phase of growth, the expression of the introduced genes for the enzymes TDC and INMT was induced. Samples were taken in intervals and diluted with acetonitrile for stopping the reaction and extracting analytes. After removal of the solid phase, samples were analyzed using LC/MS/MS with a suitable MRM method for tryptophan (Figure 2). A marker DMT is used as reference material, tryptamine (Figure 3), N-methyltryptamine (NMT)
(Figure 4) and N,N-dimethyltryptamine (DMT) (Figure 5). The method was established using authentic reference materials for each substance. Same measuring method was used for all results shown on figures 2-9. The graphs show that the machine is able to detect the reference material (Tryp. and DMT respectively). After chromatographic separation, each substance is detected using the specific retention time, a specific parent ion and the ratio of two fragments observed (Figure 10). Integration of the most selective fragment ion signal was used for quantification of the analytes. The signal correlates with the injected amount of analyte and can therefore be seen as a quantitative measure of the generated products in the samples (Figure 11). All intermediates and the final product DMT can be found in the samples 24 h after the induction of the heterologous genes. Their concentration increases over time (Figure 12 and Figure 13) while the concentration of the endogenous precursor, tryptophan, does not (Figure 14). This clearly demonstrates that the bacteria intakes the Tryptophan in the medium and the signal decreases and that the signal for DMT increases over time as tryp is depleted from the medium.
SEQUENCE LISTING
The application contains a Sequence Listing which has been submitted in ASCII format via EFS- Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on « DATE », is named « file name ».txt and is « 00000 » bytes in size. <110> Synbionik Gmbh
<120> Non-naturally occurring bacteria modified able to produce tryptophan derived compounds (DMT) <130> S75342WO
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<213> Carathanthus roseus <220>
<223> Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC)
<400> 1 atgggcagca ttgatagcac caacgtggcg atgagcaaca gcccggtggg cgaatttaaa 60 ccgctggaag cggaagaatt tcgcaaacag gcgcatcgca tggtggattt tattgcggat 120 tattataaaa acgtggaaac ctatccggtg ctgagcgaag tggaaccggg ctatctgcgc 180 aaacgcattc cggaaaccgc gccgtatctg ccggaaccgc tggatgatat tatgaaagat 240 attcagaaag atattattcc gggcatgacc aactggatga gcccgaactt ttatgcgttt 300 tttccggcga ccgtgagcag cgcggcgttt ctgggcgaaa tgctgagcac cgcgctgaac 360 agcgtgggct ttacctgggt gagcagcccg gcggcgaccg aactggaaat gattgtgatg 420 gattggctgg cgcagattct gaaactgccg aaaagtttca tgtttagcgg caccggcggc 480 ggcgtgattc agaacaccac cagcgaaagc attctgtgca ccattattgc ggcgcgcgaa 540 cgcgcgctgg aaaaactggg cccggatagc attggcaaac tggtgtgcta tggcagcgat 600 cagacccata ccatgtttcc gaaaacctgc aaactggcgg gcatttatcc gaacaacatt 660 cgcctgattc cgaccaccgt ggaaaccgat tttggcatta gcccgcaggt gctgcgcaaa 720 atggtggaag atgatgtggc ggcgggctat gtgccgctgt ttctgtgcgc gaccctgggc 780 accaccagca ccaccgcgac cgatccggtg gatagcctga gcgaaattgc gaacgaattt 840 ggcatttgga ttcatgtgga tgcggcgtat gcgggcagcg cgtgcatttg cccggaattt 900 cgccattatc tggatggcat tgaacgcgtg gatagcctga gcctgagccc gcataaatgg 960 ctgctggcgt atctggattg cacctgcctg tgggtgaaac agccgcatct gctgctgcgc 1020 gcgctgacca ccaacccgga atatctgaag aacaaacaga gcgatctgga taaagtggtg 1080 gattttaaaa actggcagat tgcgaccggc cgcaaatttc gcagcctgaa actgtggctg 1140 attctgcgca gctatggcgt ggtgaacctt cagagccata ttcgcagcga tgtggcgatg 1200 ggcaaaatgt ttgaagaatg ggtgcgcagc gatagccgct ttgaaattgt ggtgccgcgc 1260 aactttagcc tggtgtgctt tcgcctgaaa ccggatgtga gcagcctgca tgtggaagaa 1320 gtgaacaaga aactgctgga tatgctgaac agcaccggcc gcgtgtatat gacccatacc 1380 attgtgggcg gcatttatat gctgcgcctg gcggtgggca gcagcctgac cgaagaacat 1440 catgtgcgcc gcgtgtggga tctgattcag aaactgaccg atgatctgct gaaagaagcg 1500 taa 1503
<210> 2 <211 > 500
<212> PRT
<213> Carathanthus roseus <220>
<223 > Aromatic Amino acid decarboxylase enzyme
<400> 2
Met Gly Ser lie Asp Ser Thr Asn Val Ala Met Ser Asn Ser Pro Val 1 5 10 15
Gly Glu Phe Lys Pro Leu Glu Ala Glu Glu Phe Arg Lys Gin Ala His 20 25 30 Arg Met Val Asp Phe lie Ala Asp Tyr Tyr Lys Asn Val Glu Thr Tyr 35 40 45
Pro Val Leu Ser Glu Val Glu Pro Gly Tyr Leu Arg Lys Arg lie Pro 50 55 60 Glu Thr Ala Pro Tyr Leu Pro Glu Pro Leu Asp Asp He Met Lys Asp 65 70 75 80
He Gin Lys Asp He lie Pro Gly Met Thr Asn Trp Met Ser Pro Asn 85 90 95
Phe Tyr Ala Phe Phe Pro Ala Thr Val Ser Ser Ala Ala Phe Leu Gly 100 105 110
Glu Met Leu Ser Thr Ala Leu Asn Ser Val Gly Phe Thr Trp Val Ser 115 120 125
Ser Pro Ala Ala Thr Glu Leu Glu Met lie Val Met Asp Trp Leu Ala 130 135 140 Gin lie Leu Lys Leu Pro Lys Ser Phe Met Phe Ser Gly Thr Gly Gly 145 150 155 160
Gly Val He Gin Asn Thr Thr Ser Glu Ser He Leu Cys Thr He He 165 170 175
Ala Ala Arg Glu Arg Ala Leu Glu Lys Leu Gly Pro Asp Ser lie Gly 180 185 190
Lys Leu Val Cys Tyr Gly Ser Asp Gin Thr His Thr Met Phe Pro Lys 195 200 205
Thr Cys Lys Leu Ala Gly He Tyr Pro Asn Asn He Arg Leu He Pro 210 215 220 Thr Thr Val Glu Thr Asp Phe Gly He Ser Pro Gin Val Leu Arg Lys 225 230 235 240
Met Val Glu Asp Asp Val Ala Ala Gly Tyr Val Pro Leu Phe Leu Cys 245 250 255
Ala Thr Leu Gly Thr Thr Ser Thr Thr Ala Thr Asp Pro Val Asp Ser 260 265 270
Leu Ser Glu He Ala Asn Glu Phe Gly He Trp lie His Val Asp Ala 275 280 285 Ala Tyr Ala Gly Ser Ala Cys lie Cys Pro Glu Phe Arg His Tyr Leu 290 295 300
Asp Gly lie Glu Arg Val Asp Ser Leu Ser Leu Ser Pro His Lys Trp 305 310 315 320 Leu Leu Ala Tyr Leu Asp Cys Thr Cys Leu Trp Val Lys Gin Pro His 325 330 335
Leu Leu Leu Arg Ala Leu Thr Thr Asn Pro Glu Tyr Leu Lys Asn Lys 340 345 350
Gin Ser Asp Leu Asp Lys Val Val Asp Phe Lys Asn Trp Gin He Ala 355 360 365
Thr Gly Arg Lys Phe Arg Ser Leu Lys Leu Trp Leu He Leu Arg Ser 370 375 380
Tyr Gly Val Val Asn Leu Gin Ser His lie Arg Ser Asp Val Ala Met 385 390 395 400 Gly Lys Met Phe Glu Glu Trp Val Arg Ser Asp Ser Arg Phe Glu He 405 410 415
Val Val Pro Arg Asn Phe Ser Leu Val Cys Phe Arg Leu Lys Pro Asp 420 425 430
Val Ser Ser Leu His Val Glu Glu Val Asn Lys Lys Leu Leu Asp Met 435 440 445
Leu Asn Ser Thr Gly Arg Val Tyr Met Thr His Thr He Val Gly Gly 450 455 460
He Tyr Met Leu Arg Leu Ala Val Gly Ser Ser Leu Thr Glu Glu His 465 470 475 480 His Val Arg Arg Val Trp Asp Leu lie Gin Lys Leu Thr Asp Asp Leu 485 490 495
Leu Lys Glu Ala 500 <210> 3
<211 > 792 <212> DNA <213> Homo sapiens
<220>
<223> Indolethylamine N-methyltransferase (GNMT) (EC Nr. : 2.1.1.49 (formerly EC Nr. : 2.1.1.81))
<400> 3 atgaaaggcg gctttaccgg cggcgatgaa tatcagaaac attttctgcc gcgcgattat 60 ctggcgacct attatagctt tgatggcagc ccgagcccgg aagcggaaat gctgaaattt 120 aacctggaat gcctgcataa aacctttggc cctggcggcc ttcagggcga taccctgatt 180 gatattggca gcggcccgac catttatcag gtgctggcgg cgtgcgatag ctttcaggat 240 attaccctga gcgattttac cgatcgcaac cgcgaagaac tggaaaaatg gctgaagaaa 300 gaaccgggcg cgtatgattg gaccccggcg gtgaaatttg cgtgcgaact ggaaggcaac 360 agcggtcgct gggaagaaaa agaagaaaaa ctgcgcgcgg cggtgaaacg cgtgctgaaa 420 tgcgatgtgc atctgggcaa cccgctggcg cctgcggtgc tgccgctggc ggattgcgtg 480 ctgaccctgc tggcgatgga atgcgcgtgc tgtagcctgg atgcgtatcg cgcggcgctg 540 tgcaacctgg cgagcctgct gaaaccgggc ggccatctgg tgaccaccgt gaccctgcgc 600 ctgccgagct atatggtggg caaacgcgaa tttagctgcg tggcgctgga aaaagaagaa 660 gtggaacagg cggtgctgga tgcgggcttt gatattgaac agctgctgca tagcccgcag 720 agctatagcg tgaccaacgc ggcgaacaac ggcgtgtgct ttattgtggc gcgcaagaaa 780 ccgggcccgt aa 792
<210> 4
<211 > 263
<212> PRT
<213> Homo sapiens
<220>
<223> Indolethylamine N-methyltransferase (GNMT) (EC Nr. : 2.1.1.49 (formerly EC Nr. : 2.1.1.81))
<400> 4
Met Lys Gly Gly Phe Thr Gly Gly Asp Glu Tyr Gin Lys His Phe Leu 1 5 10 15
Pro Arg Asp Tyr Leu Ala Thr Tyr Tyr Ser Phe Asp Gly Ser Pro Ser 20 25 30
Pro Glu Ala Glu Met Leu Lys Phe Asn Leu Glu Cys Leu His Lys Thr 35 40 45
Phe Gly Pro Gly Gly Leu Gin Gly Asp Thr Leu lie Asp lie Gly Ser 50 55 60 Gly Pro Thr He Tyr Gin Val Leu Ala Ala Cys Asp Ser Phe Gin Asp 65 70 75 80
He Thr Leu Ser Asp Phe Thr Asp Arg Asn Arg Glu Glu Leu Glu Lys 85 90 95
Trp Leu Lys Lys Glu Pro Gly Ala Tyr Asp Trp Thr Pro Ala Val Lys 100 105 110
Phe Ala Cys Glu Leu Glu Gly Asn Ser Gly Arg Trp Glu Glu Lys Glu 115 120 125 Glu Lys Leu Arg Ala Ala Val Lys Arg Val Leu Lys Cys Asp Val His 130 135 140
Leu Gly Asn Pro Leu Ala Pro Ala Val Leu Pro Leu Ala Asp Cys Val 145 150 155 160 Leu Thr Leu Leu Ala Met Glu Cys Ala Cys Cys Ser Leu Asp Ala Tyr 165 170 175
Arg Ala Ala Leu Cys Asn Leu Ala Ser Leu Leu Lys Pro Gly Gly His 180 185 190
Leu Val Thr Thr Val Thr Leu Arg Leu Pro Ser Tyr Met Val Gly Lys 195 200 205
Arg Glu Phe Ser Cys Val Ala Leu Glu Lys Glu Glu Val Glu Gin Ala 210 215 220
Val Leu Asp Ala Gly Phe Asp lie Glu Gin Leu Leu His Ser Pro Gin 225 230 235 240 Ser Tyr Ser Val Thr Asn Ala Ala Asn Asn Gly Val Cys Phe lie Val 245 250 255
Ala Arg Lys Lys Pro Gly Pro 260

Claims

Claims
1. A non-naturally occurring microorganism modified to produce N,N-Dimethyltryptamine.
2. A non-naturally occurring microorganism according to claim 1 expressing the enzymes: a. Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) (EC Nr.: 4.1.1.28) b. Indolethylamine N-methyltransferase (INMT) (EC Nr. : 2.1.1.49)'
3. A non-naturally occurring microorganism according to claim 1 or 2 expressing a. the Aromatic Amino acid decarboxylase enzyme is a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 2 and wherein the Aromatic Amino acid decarboxylase enzyme retains Tryptamine 4-monooxygenase activity, and b. the Indolethylamine N-methyltransferase (INMT) enzyme is a polypeptide having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO 4 and wherein the Indolethylamine N-methyltransferase retains Isoform of Indolethylamine N-methyltransferase (INMT) enzyme activity.
4. A non-naturally occurring microorganism according to any of claims 1 to 3 comprising a. a nucleic acid sequence encoding Tryptophan Decarboxylase where in the sequence of the nucleic acid encoding Tryptophan Decarboxylase is at least 80% identical to nucleic acid sequence of SEQ ID No. 1. b. a nucleic acid sequence encoding Indolethylamine N-methyltransferase where in the sequence of the nucleic acid encoding Indolethylamine N-methyltransferase is at least 80% identical to the nucleic acid sequence of SEQ ID No. 3.
5. A non-naturally occurring microorganism according to claim 4 comprising an inducible promoter operably linked to the nucleic acid encoding Tryptophan Decarboxylase and/or to the nucleic acid encoding Indolethylamine N-methyltransferase.
6. A non-naturally occurring microorganism according any of claims 1-5 modified to produce 5-MeOH- N,N-Dimethyltryptamine
7. A non-naturally occurring microorganism according to any of claims 1-6 wherein the
5 microorganism is optimized for a higher production of Tryptophan.
8. A non-naturally occurring microorganism according to any of claims 1-7 wherein the microorganism is able to grow in ammonium minimal medium. io
9. A non-naturally occurring microorganism according to any of the preceding claims wherein the microorganism is a bacterium or a yeast.
10. A non-naturally occurring microorganism according to claim 9 wherein the bacterium is selected from the genus Escherichia, Sccharomyces, Clostridium, Bacillus, Lactococcus, is Zymomonas, Corynebacterium, Pichia, Candida, Hansenula, Trichoderma,
Acetobacterium, Ralstonia, Cupravidor, Salmonella, Klebsiella, Paenibacillus, Lactobacillus, Rhodococcus, Enterococcus, Alkaligenes, Brevibacterium, Methylobacterium, Methylobacterium, Methylococcus, Methylomonas, Methylocystis and Methylosinus.
20
11. A non-naturally occurring microorganism according to claim 10 wherein the bacterium is selected from the genus Escherichia and Zymomonas.
12. A non-naturally occurring microorganism according to claim 11 wherein the bacterium is 25 Escherichia coli or Zymomonas mobilis.
13. Method of converting L-Tryptophan and/or Tryptamine into DMT and/or 5-Me-OH- DMT, the method comprising culturing non-naturally occurring microorganism of any of claims 1-12 in a suitable culture medium under conditions suitable to induce expression of
30 Tryptophan Decarboxylase (Aromatic Amino acid Decarboxylase, AADC) and /or Indolethylamine N-methyltransferase, and then harvesting the cultured cells or spent medium, thereby converting -Tryptophan and/or Tryptamine into DMT and/or 5-Me-OH- DMT.
14. Method of claim 13 further comprising the step of : a) lysing the non-naturally occurring microorganism b) purifying DMT and/or 5-Me-OH-DMT from the cell lysate, thereby producing a purified target metabolic product.
15. Method of claim 14 further comprising formulating DMT or 5-Me-OH-DMT in a pharmaceutical composition.
PCT/EP2020/084773 2019-12-04 2020-12-04 Non-naturally occurring bacteria modified able to produce tryptophan derived compounds WO2021110992A1 (en)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2023130075A3 (en) * 2021-12-31 2023-09-28 Empyrean Neuroscience, Inc. Genetically modified organisms for producing psychotropic alkaloids
WO2023130191A1 (en) * 2022-01-10 2023-07-13 Core One Labs Inc. Production of psychedelic compounds

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