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WO2025068602A2 - Production and medical use of psilocybin and related compounds - Google Patents

Production and medical use of psilocybin and related compounds Download PDF

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Publication number
WO2025068602A2
WO2025068602A2 PCT/EP2024/077495 EP2024077495W WO2025068602A2 WO 2025068602 A2 WO2025068602 A2 WO 2025068602A2 EP 2024077495 W EP2024077495 W EP 2024077495W WO 2025068602 A2 WO2025068602 A2 WO 2025068602A2
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yeast cell
cell
gene
recombinant yeast
psilocybin
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WO2025068602A3 (en
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Evaldas ČIPLYS
Laura Martinkute KORSAKOVA
Eimantas ŽITKUS
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Psylink Uab
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Psylink Uab
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Definitions

  • the present disclosure relates to novel production process for psilocybin and related compounds as well as products related to the production process, including recombinant cells in which these compounds can be produced.
  • the present disclosure relates to novel compounds related to psilocybin, which may be produced by the production processes, and their use in medicine.
  • Psilocybin is a naturally occurring compound of the alkaloid class, found in some species of fungi, in particular members of the genus Psilocybe, e.g. P. mexicana, P. cubensis, P.
  • Psilocybin is a prodrug; in the body psilocybin (3-[2-dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate is dephosphorylated to psilocin (4-hydroxy-N,N-dimethyltryptamine), which is a hallucinogenic compound.
  • psilocybin 3-[2-dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate is dephosphorylated to psilocin (4-hydroxy-N,N-dimethyltryptamine), which is a hallucinogenic compound.
  • the hallucinogenic effects of mushrooms containing psilocybin have long been known, and psilocybin was isolated from mushrooms in the 1950s.
  • Psilocin has a similar chemical structure to serotonin (5-hydroxytryptamine), an important neurotransmitter in the human body, and is known to bind to different human serotonin receptors including the 5-hydroxytryptamine 2A (5-HT2A) receptor.
  • 5-HT2A 5-hydroxytryptamine 2A
  • the present invention provides a method for producing a target alkaloid from a precursor via a metabolic pathway in a recombinant yeast cell, wherein the metabolic pathway comprises a psiH enzyme, a psiK enzyme and a psiM enzyme, the method comprising culturing the recombinant yeast cell in a culture medium comprising the precursor under conditions suitable to allow the precursor to enter the recombinant yeast cell such that the recombinant yeast cell produces the target alkaloid, wherein the precursor is a substrate for the psiH enzyme.
  • the present invention further provides a recombinant yeast cell comprising a psiH gene, a psiK gene, and a psiM gene, wherein the recombinant yeast cell: (i) does not comprise an L- tryptophan decarboxylase gene; and/or (ii) comprises an exogenous CPR gene and an adenosylhomocysteinase (SAH1) gene.
  • the present invention provides a yeast cell culture produced by the method of the invention described above and comprising the recombinant yeast cell and the target alkaloid. Further provided is a medical formulation comprising the recombinant yeast cells containing the target alkaloid and at least one excipient.
  • the present invention provides a method for extracting a produced target alkaloid comprising an amine group and an -HPO 4 group from a recombinant yeast cell, the method comprising: (i) contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell; (ii) centrifuging the extraction mixture to form a supernatant comprising the target alkaloid; (iii) ensuring the supernatant has a pH between 10 and 14 and has a low ionic strength solution such that the charge of N in the amine group is neutral; (iv) applying the supernatant to an anion exchange column; and (v) using a hydroxyl ion gradient to elute at least one fraction comprising the target alkaloid.
  • the amine group is selected from -N(CH 3 ) 2 and an -NHCH 3 group.
  • a yeast cell extract prepared by this method is also provided.
  • the present invention provides a method for increasing methylation capacity of a psiM enzyme in a recombinant yeast cell, the method comprising increasing expression of an adenosylhomocysteinase (SAH1) enzyme in the recombinant yeast cell so as to increase the capacity of the recombinant yeast cell to hydrolyse S-adenosyl-l-homocysteine.
  • SAH1 adenosylhomocysteinase
  • the present invention provides a compound according to Formula (I), or a pharmaceutically acceptable salt thereof, wherein: X is S, O, C, or N, wherein when X is S or O, R 8a and R 8b are absent, and wherein when X is N, R 8b is absent, Z is C or N, wherein when Z is N, R 2 is absent, and wherein when X is N, Z is N, R 4 is -HPO 4 , or -OH, Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3 , R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3 , R 2 , R 3a , R 3b , R 3c , R 3d , R 8a and R 8b are independently selected from H, or D.
  • X is S, O, C, or N
  • R 8a and R 8b are absent
  • Z is C or N
  • the present invention provides a compound according to Formula (IA): wherein: R 4 is -HPO4, or -OH, Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3 , R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3 , R 2 , R 3a , R 3b , R 3c , R 3d are independently selected from H, or D.
  • R 2 , R 3a , R 3b , R 3c , R 3d , R 5 , R 6 , R 7 are all H.
  • R 4 is -HPO 4 , or -OH
  • Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3
  • R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3
  • R 2 , R 3a , R 3b , R 3c , R 3d are independently selected from H, or D.
  • R 2 , R 3a , R 3b , R 3c , R 3d are all H.
  • the target alkaloids according to formula IA and IB may also be produced by the methods of the invention and may be used in the products and compositions of the invention.
  • the present invention further provides a pharmaceutical composition comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the present invention provides the compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in medicine.
  • the present invention provides improved methodology for the production of target alkaloids, and especially psilocybin, psilocin and related compounds in recombinant yeast cells.
  • the method described herein advantageously avoids the need to rely on endogenously generated L-tryptophan as the first substrate for the metabolic pathway and instead begins with a later substrate (also referred to herein as precursor) that is the substrate for the psiH enzyme.
  • a later substrate also referred to herein as precursor
  • suitable conditions can be obtained that allow this later substrate in the pathway to be efficiently taken up by the cells.
  • the methods described herein can be used to obtain superior conversion rates and titres of the target alkaloid.
  • the present invention provides purification methods that advantageously lead to the efficient and effective purification of the target alkaloids.
  • the purification methods can be used to avoid the need for cell disruption and chromatographic separation steps.
  • Figure 1 provides a schematic of one example of the biosynthetic route of psilocybin production according to present invention.
  • Figure 2 is a schematic showing the vector map for pPpGAP-KanS-HIS4-psiM (P. cyan.)-psiK (P. cub.)-psiH (P. cyan.)-CPR (P. cyan.)-SAH1 (P. p.).
  • Figure 3 is a picture showing SDS-PAGE analysis of selected P.pastoris strains having psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus and an additional copy of endogenous P. pastoris SAH1 (Adenosylhomocysteinase) gene integrated into the genome.
  • Figure 4 provides results of HPLC analysis of an extract of yeast cells converting tryptamine to psilocybin.
  • Figure 5 provides a chromatogram of the first step of psilocybin purification using 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 6 provides a chromatogram of the second step of psilocybin purification using 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 7 provides HPLC analysis of psilocybin product at 230 nm of combined 22-32 fractions after second step purification using Sepharose HP.
  • Figures 8A and 8B provide analysis showing production of Substance (4) from Example 2.
  • Figure 8A provides a chromatogram from HPLC analysis showing production of Substance (4).
  • Figure 8B provides a mass spectrum for Substance (4).
  • Figures 9A and 9B provide analysis showing production of Substance (5) from Example 2.
  • Figure 9A provides a chromatogram from HPLC analysis showing production of Substance (5).
  • Figure 8B provides a mass spectrum for Substance (5).
  • Figure 10 provides a chromatogram of Substance (5) from Example 2 purification, using 150 ml volume (XK 26/40) SepharoseQ HP.
  • Figures 11A-C provide chromatograms from HPLC analysis of whole cell broth (WCB) extract after addition of 2mM substrate – 2- (Benzofuran-3-yl) ethylamine (Substance (1) Example 3).
  • Figure 11A shows result 5 mins after addition.
  • Figure 11B shows result 4 hours after addition.
  • Figure 11C shows result 20 hours after addition.
  • Figure 12 provides a chromatogram of the first step of O-Psilocybin purification (in Example 3) using a 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 13 provides a chromatogram of the second step of O-Psilocybin purification (in Example 3) using a 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 14 provides HPLC analysis of combined 11-13 fractions after the second step purification of O-Psilocybin (in Example 3) using Sepharose HP.
  • Figure 15 provides bar charts showing the dose-response for compounds tested in the 5-HT2A serotonin receptor agonist assay. Cells were treated with the test compounds at serial concentrations. Data points represent the mean ⁇ SD for each condition for a single experiment performed in triplicate. Results are expressed as the fluorescence intensity (arbitrary units, a.u.) of Fluo-4. C- represents vehicle.
  • Figure 16 provides the dose response curve for PSY-1185 tested in the 5-HT2A serotonin receptor agonist assay. Data is presented as the percentage of activity of the HTR2A normalized against the zero control (vehicle) that represents the 0% of activity and the maximum concentration of each compound that represents the 100%. DETAILED DESCRIPTION As noted above, the biosynthetic pathway for the production of psilocybin was discovered in 2017 by a group of scientists at the Leibniz Institute for Natural Product Research and Infection Biology (Fricke et al., September 25, 2017, Angewandte Chemie, 56(40): 12352-12355).
  • This route (which is shown in Figure 1) uses the enzymes psiD, psiH, psiK, and psiM, to produce psilocybin from the precursor tryptophan; tryptophan being produced in the cell via other metabolic pathways, e.g. the Shikimate-Chorismate pathway as described in Milne et al., (Metabolic Engineering, 60 (2020): 25-36). Further work in this area has been done by Vogan et al., (US 11,441164 B2, published 13 September 2022), which suggested the construction of S. cerevisiae yeast strains that have been genetically engineered to increase the metabolic flux towards L-tryptophan.
  • the present inventors have found that it is possible to improve the production of the target alkaloid by starting the metabolic pathway with a precursor that is a substrate for the psiH enzyme (as shown in Figure 1), since suitable conditions can be created in the culture media that allow the precursor to be efficiently taken up by the yeast cells.
  • This route is advantageous as it is not limited to cell-produced tryptophan.
  • the target alkaloid may be according to formula (I): in which X is N, S, O or C, wherein when X is N, R 8b is absent, and wherein when X is S or O, R 8a and R 8b are absent, Z is C or N, wherein when Z is N, R 2 is absent, R 4 is -HPO 4 , or OH, Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3 , R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3 , R 2 , R 3a , R 3b , R 3c , R 3d , R 8a and R 8b are independently selected from H or D.
  • formula (I) in which X is N, S, O or C, wherein when X is N, R 8b is absent, and wherein when X is S or O, R 8a and R 8b are absent, Z is
  • the precursor can be selected based on the target alkaloid and the reactions that the enzymes psiH, psiK and psiM catalyse in the metabolic pathway. While these enzymes have been shown to produce psilocybin (as shown in one example of the invention in Figure 1), the examples provided herein surprisingly show that the enzymes are able to accommodate variations in their substrates (in particular in locations on the substrate molecules that are away from positions involved in the enzymatic reaction), allowing the method described herein to be used to produce compounds that are related to psilocybin.
  • X is N and Z is C, and more preferably the target alkaloid is psilocybin.
  • the precursor can be tryptamine.
  • the precursors can be 2-(benzothien-3-yl)ethylamine, 2- (Benzofuran-3-yl)ethylamine and 2-(1H-inden-3-yl)ethanamine, respectively.
  • the experimental work described herein shows that the alteration of the substrates of psiH, psiK and psiM enzymes does not prevent these from catalysing the reactions.
  • X is N
  • Z is N and R 2 is absent
  • R 3a , R 3b , R 3c , R 3d , R 5 , R 6 , R 7 ,R 8a and R 8b are all H.
  • the precursor can be 2-(1H-indazol-3- yl)ethanamine hydrochloride.
  • R 5 , R 6 , R 7 are independently selected from H, and D, and are more preferably H.
  • Y 1 is -N + H(CH 3 ) 2 .
  • the enzymes that may be used in the method of the present invention are as follows: PsiH Enzyme -
  • the psiH enzyme is a tryptamine 4-monooxygenase, which is able to catalyse the conversion of tryptamine to 4-hydroxytryptamine.
  • the psiH enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens.
  • the psiH enzyme may be produced from a cDNA sequence having SEQ ID NO: 7.
  • the psiH enzyme comprises an amino acid sequence having SEQ ID NO: 8, or an amino acid sequence having at least 85% identity to SEQ ID NO: 8. More preferably the psiH enzyme comprises an amino acid sequence having SEQ ID NO: 8, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 8.
  • PsiK Enzyme – The psiK enzyme is a 4-hydroxytrptamine kinase, which is able to catalyse the conversion of 4-hydroxytryptamine to norbaeocystin.
  • the psiK enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens.
  • the psiK enzyme may be produced from a cDNA sequence having SEQ ID NO: 3.
  • the psiK enzyme comprises an amino acid sequence having SEQ ID NO: 4, or an amino acid sequence having at least 85% identity to SEQ ID NO: 4. More preferably the psiK enzyme comprises an amino acid sequence having SEQ ID NO: 4, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 4.
  • the psiM enzyme is a methyltransferase, which is able to catalyse the methylation of norbaeocystin to form baeocystin, and the subsequent methylation of baeocystin to form psilocybin.
  • the psiM enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens.
  • the psiM enzyme may be produced from a cDNA sequence having SEQ ID NO: 5.
  • the psiM enzyme comprises an amino acid sequence having SEQ ID NO: 6, or an amino acid sequence having at least 85% identity to SEQ ID NO: 6.
  • the psiM enzyme comprises an amino acid sequence having SEQ ID NO: 6, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 6.
  • Cytochrome P450 reductase (CPR) – a CPR enzyme may also be used in the method of the invention.
  • yeast endogenous P450 reductases are not compatible with psiH. Accordingly, it is preferred to use a recombinant yeast cell expressing an exogenous CPR gene in order to increase psiH efficiency.
  • the CPR enzyme may be derived from Psilocybe cyanesens, and may be produced from a nucleotide sequence having SEQ ID NO: 1.
  • the CPR enzyme comprises an amino acid sequence having SEQ ID NO: 2, or an amino acid sequence having at least 85% identity to SEQ ID NO: 2. More preferably the CPR enzyme comprises an amino acid sequence having SEQ ID NO: 2, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 2.
  • Adenosylhomocysteinase (SAH1) – the SAH1 enzyme may also be used in the method of the invention to improve the efficiency of the psiM methylation steps.
  • the present inventors have surprisingly found that the S-adenosyl-L-homocysteine (SAH), the molecule that is formed when a methyl group is transferred from SAM to a target molecule, acts as competitive inhibitor of the SAM-dependent methyl transferase reactions catalysed by psiM.
  • SAH S-adenosyl-L-homocysteine
  • the yeast cell may already comprise an endogenous SAH1 gene. However, one or more (additional) copies of a SAH1 gene may be incorporated into the recombinant yeast cell.
  • the gene may be derived from P. pastoris, and may comprise the nucleotide sequence of SEQ ID NO: 9.
  • the SAH1 enzyme comprises an amino acid sequence having SEQ ID NO: 10, or an amino acid sequence having at least 85% identity to SEQ ID NO: 10. More preferably the SAH1 enzyme comprises an amino acid sequence having SEQ ID NO: 10, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO:
  • the recombinant yeast cell used in the method of the invention does not comprise a psiD gene or other gene encoding a tryptophan decarboxylase.
  • a further aspect of the invention relates to a method for increasing methylation capacity of a psiM enzyme in a recombinant yeast cell.
  • the method comprises increasing expression of an adenosylhomocysteinase (SAH1) enzyme in the recombinant yeast cell so as to increase the capacity of the recombinant yeast cell to hydrolyse S-adenosyl-l-homocysteine.
  • SAH1 adenosylhomocysteinase
  • expression may be increased by transforming the yeast cell with one or more copies of an SAH1 gene encoding the SAH1 enzyme; and/or increasing transcription from an SAH1 gene encoding the SAH1 enzyme using a promoter or an enhancer.
  • a method may be used as part of a method for producing a target alkaloid from a precursor as described above. Alternatively, it may be used in combination with the psilocybin production methods already described in the prior art.
  • the psiM enzyme carries out two methylation reactions to produce a target alkaloid in which Y 1 is -N + H(CH 3 ) 2 , however, by adding more substrate and/or by restricting the methylation capacity of the psiM enzyme a target alkaloid in which Y 1 is -N + H 2 CH 3 can be produced.
  • the present disclosure provides recombinant yeast cells that can be used in the methods described herein to produce the target alkaloid. Types of yeast cell that are suitable for performing the method of the invention are those already know in the art for recombinant protein production.
  • the yeast may be from the genus Pichia, such as Pichia pastoris, from the genus Sacchromyces, such as S. cerevisiae or S. pombe, or from the genus Yarrowia such as Y. lipolytica.
  • the recombinant yeast cell is from the genus Pichia, and most preferably the yeast cell is Pichia pastoris.
  • Recombinant vectors carrying the genes for the enzymes described above can be transformed into the yeast cell by methods known in the art. It is preferred that the DNA constructs are integrated into the yeast genome in order to provide stable expression of the genes.
  • the genes for the enzymes described above can be placed under the control of inducible or constitutive promoters or gene expression control elements.
  • Suitable promoters are known in the art for the production of recombinant proteins in yeast. However, where the yeast is Pichia, it is preferred that the promoters AOX1 or GAP are used. The GAP promoter is most preferred.
  • the recombinant yeast cell is cultured in a culture medium comprising the precursor under conditions that are suitable to allow the precursor for the enzyme psiH to enter the recombinant host cells.
  • the precursors for the enzyme psiH are charged molecules in normal yeast growth conditions, which do not effectively cross the yeast cell membrane.
  • the present inventors have surprisingly found that they are able to culture the recombinant yeast cells at a pH of 6 to 7.8, preferably at a pH of 7.5 to 7.8 and most preferably at a pH of 7.8.
  • these pH values are not particularly close to the pKa of the precursor molecules (with the pKa of tryptamine being approximately 10.2 at which yeast cannot be readily cultivated)
  • the present inventors have observed rapid diffusion of the precursor into the yeast cell, as a first step to efficient production of the target alkaloid.
  • a suitable phosphate source in the culture media is therefore sodium hexametaphosphate.
  • the precursor may be added to the culture media in batches, and one batch may be added after the full conversion of the previously added precursor has been achieved.
  • the constant addition of precursor may lead to an accumulation of intermediates.
  • the precursor is added to the culture media at a rate of 4 to 6 ⁇ M every 2.5 to 3.5 hours per 1 gram wet cell weight.
  • the method according to the present invention can achieve advantageous production rates of the target alkaloid.
  • the target alkaloid can be produced at a rate of at least 1 g/litre of culture every 24 hours, preferably at least 3 g/litre of culture every 24 hours, more preferably at least 5g/litre of culture every 24 hours.
  • the present invention provides a method for extracting a produced target alkaloid comprising an amine group and an -HPO 4 group from a recombinant yeast cell, the method comprising: (i) contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell; (ii) centrifuging the extraction mixture to form a supernatant comprising the target alkaloid; (iii) ensuring the supernatant has a pH between 10 and 14 and has a low ionic strength solution such that the charge of N in the -N(CH 3 ) 2 group is neutral; (iv) applying the supernatant to an anion exchange column; and (v) using a hydroxyl ion gradient to elute at least one fraction comprising the target alkaloid.
  • the amine group may be an -N(CH 3 ) 2 group or an -NHCH 3 group.
  • step (iii) would normally include a step of buffer exchange, e.g. via nanofiltration, to transfer the target alkaloid into a suitable buffer so that it can bind to the anion exchange column.
  • the whole cell broth may be centrifuged to separate the yeast cells from the growth media and the yeast cells washed in order to remove the media and placed the cells in a suitable media prior to contact with the acetonitrile.
  • the solution for extraction in (i) may comprise 15 to 75% (v/v) acetonitrile, preferably 20 to 60% (v/v) acetonitrile, more preferably 25 to 35% (v/v) acetonitrile.
  • the supernatant has a pH between 10 and 14 and a low ionic strength such that the charge of the N in the -N(CH 3 ) 2 group is neutral.
  • Such low ionic strength solution is a solution containing 0.05mM to 50 mM ions wherein the ions have a lower selectivity towards the positively charged groups of the anion exchange column than the -HPO 4 group.
  • the anion exchange column may be a Sepharose column, e.g. Sepharose Q HP, Sepharose Q, or Sepharose DEAE.
  • a hydroxyl ion gradient is used to elute at least one fraction comprising the target alkaloid.
  • a hydroxyl ion gradient has been found by the inventors to produce the best selectivity for the target alkaloid.
  • the at least one fraction may be two or more fractions and the method may comprise combining the two or more fractions, diluting the combined fractions and reapplying to the anion exchange column, and repeating step (v).
  • the present invention provides novel compounds related to psilocybin and to psilocin. These compounds can be prepared by the methods described above.
  • Substance (6) (A dephosphorylated form of the product is shown as Substance (6) (“S-psilocin”) below.) Substance 1 Name (IUPAC) 2-(benzothien-3-yl)ethylamine hydrochloride Substance 2 Substance 5 Formula C12H16NO4PS MW 301.2988 Fermentation technology a) Collection of yeast P. pastoris strains with integrated into the genome psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus having different S-tryptamine (Substance (1)) to S-psilocybin (Substance (5)) conversion properties were screened to find a strain with the best conversion rate.
  • Substance 1 Name
  • IUPAC 2-(benzothien-3-yl)ethylamine hydrochloride
  • Substance 2 Substance 5 Formula C12H16NO4PS MW 301.2988 Fermentation technology a) Collection of yeast
  • Target genes were placed under strong constitutive GAP promoter (enzymes are produced constantly) or very strong inducible AOX1 promoter (enzymes are produced only when methanol is added). Our research has shown that currently the best option is P. pastoris strain, where target genes are placed under GAP promoter.
  • CBS7435 strain CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1; CBS7435_pPpGAP- HIS4-psiM-psiK-psiH-CPR_2, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH- CPR_5, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_8. II.
  • Glycerol 98% 20 g Milli Q ultrapure water (UPW) 912 g Calcium sulfate dihydrate 0.46 g Magnesium sulfate heptahydrate 5.84 g Potassium sulfate 7.34 g Ammonium sulfate 9.0 g Hexametaphosphate 300g/L solution 84 ml Trace metal solution (PTM) 4 ml Table 3 – BSM media, 1 Litre Copper (II) sulfate 3.0 g Sodium iodide 0.040 g Manganese (II) sulfate 1.50 g Sodium molybdate 0.100 g Boric acid 0.010 g Cobalt (II) chloride 0.250 g Zinc chloride 10.0 g Iron (II) sulfate heptahydrate 32.5 g Biotin 0.100 g Sulfuric acid, conc.
  • pastoris fermentation conditions that allow uptake of S-tryptamine to the cell, and efficient conversion to S-psilocybin were developed, that allow complete conversion of 2mM of S-tryptamine to S-psilocybin resulting in 600 mg of S-psilocybin in 1L WCB.
  • the process uses BSM media as shown below in Table 5.
  • Glycerol 98% 63 g Milli Q ultrapure water (UPW) 912 g Calcium sulfate dihydrate 0.46 g Magnesium sulfate heptahydrate 5.84 g Potassium sulfate 7.34 g Ammonium sulfate 9.0 g Hexametaphosphate 300g/L solution 84 ml Trace metal solution (PTM) 4 ml Table 5 – BSM media, 1L Bioreactors: EDF-1.2 bioreactors (Biotechniskais centrs, AS) Starting fermentation conditions: 28 ⁇ C, 1 vvm air, pH 5.0, 400 rpm.
  • S-psilocybin 600 mg S-psilocybin in 1L of WCB. About 20-25% of S-psilocybin is in the culture media and the rest is inside the yeast cells.
  • pH 5.0 S-tryptamine is relatively uncharged molecular that can’t freely cross yeast cells plasma membrane.
  • S- tryptamine is partly charged and can cross plasma membrane.
  • Most of the phosphate salts used for yeast fermentation precipitate under pH 7 – 7.8.
  • HMP sodium hexametaphosphate
  • S-tryptamine conversion to S-psilocybin is 10 ⁇ M of S- tryptamine to S-psilocybin in 6-7 hours per 1 gram of wet cell weight (WCW) by CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1 strain. Average amount of WCW during conversion is about 200-240 g/L, so conversion rate is 2 mM of S-tryptamine to 2 mM S- psilocybin per 6-7 hours (600 mg of S-psilocybin is synthesized in 6-7 hours).
  • S-psilocybin Different pH values, glycerol feeding profiles, amount and addition time of substrate Substance (1) - (S-tryptamine) were analyzed to develop the optimal fermentation conditions for efficient and complete conversion of substrate to final product - Substance (5) (S-psilocybin). Purification technology Testing was performed to achieve efficient and cost-effective extraction and purification of S- psilocybin. Materials and methods used for S-psilocybin extraction optimization: I. Yeast cell biomass after fermentation containing 1,5-2 mg of S-psilocybin per gram of WCW. II. S-psilocybin was extracted by resuspending cells in 1:1 to 1:5 g of WCW to ml of solution ratio using different solutions. Mixture was vortexed for 2 min.
  • Extracts were analyzed by HPLC. Materials and methods used for S-psilocybin chromatographic purification optimization: I. Extract of yeast cells, containing S-psilocybin, using 30% of acetonitrile in 1mM NaOH. II. Classical chromatography techniques were used to determine optimal S-psilocybin binding conditions, binding capacity and elution profiles. III. Fractions were analyzed by HPLC. It was noted that the findings described above in Example 1 in relation to the extraction and purification of psilocybin also applied to S-psilocybin.
  • Example 1 the purification methodology points 1 to 17 set out in Example 1 can also be used.
  • S-psilocybin elutes as a single peak at about 70-75 mM NaOH 12.7-12.9 mS/cm ( Figure 10, fractions 33-34).
  • This Example demonstrates the ability of the method of the invention to produce target alkaloids that are related to psilocybin.
  • Example 3 Biosynthesis of 3-(N,N-dimethylaminoethyl)benzo[b]furan-4-yl phosphate (O-Psilocybin) in the yeast P.
  • Substance 6 (A dephosphorylated form of the product is shown as Substance 6 (“O-psilocin”).)
  • Substance 1 Name (IUPAC): 2-(benzofuran-3-yl)ethylamine hydrochloride Formula: C10H11NO*ClH MW: 197.664 CAS no. 27404-32-6
  • Substance 2 Name 3-(2-aminoethyl)-1-benzofuran-4-ol Formula: C10H11NO2 MW: 177.20 CAS no. 1890736-76-1
  • Substance 4 Substance 5
  • Substance 6 Fermentation technology a) Yeast P.
  • Example 4 Steps for biosynthesis of further compounds in the yeast P. pastoris cells using enzymes from fungi of Psilocybe genus
  • Example 4A To provide a compound according to formula I described herein where X is N and Z is C and W is N (a benzimidazole derivative).
  • the substrate can be 2- (1H-Benzimidazol-1-yl)ethylamine hydrochloride and the following synthesis pathway can be used: 1. Substrate (starting material) 2-(1H-Benzimidazol-1- yl)ethylamine hydrochloride 2. 3. 4. 5. 6. Example 4B — To provide a compound according to formula I described herein where X is O and Z is N and W is C (a benzisoxazole derivative).
  • the substrate (starting material) can be 1,2-Benzisoxazole-3-ethanamine and the following synthesis pathway can be used: Example 4C – To provide a compound according to formula I described herein where X is O and Z is C and W is N (a benzo[d]oxazole derivative).
  • the substrate (starting material) can be 2-(benzo[d]oxazol-3(2H)-yl)ethan-1-amine and the following synthesis pathway can be used: FUNCTIONAL STUDIES
  • the functional activity of the psilocybin related compounds described herein can be shown in vitro using radioligand binding assays and in vivo using head twitch response studies.
  • Competition Assays can be used to show binding to 5-HT receptors, e.g. 5-HT1A.
  • a suitable example assay is described by Gifford Bioscience Limited (www.giffordbioscience.com - Radioligand Binding Assay Protocols). This assay is as follows: Membrane preparation: Frozen tissue or washed cells are homogenized in 20 volumes of cold lysis buffer (50mM Tris-HCl, 5 mM MgCl2, 5 mM EDTA, protease inhibitor cocktail). After a low speed spin (100 x g for 3 minutes) to remove large tissue chunks (tissue homogenates), the homogenate is centrifuged at 17,000 x g for 10 minutes at 4 °C to pellet the membranes.
  • the pellet is resuspended in fresh buffer and centrifuged at the same speed for a second time, again at 4 °C.
  • the pellet is then resuspended into buffer (15 ml) containing 10% sucrose as a cryoprotectant, divided into 1 ml aliquots and stored at -80 °C.
  • a sample of the homogenate is analyzed for protein content.
  • the membrane preparation is thawed and the pellet resuspended in final assay binding buffer (50 mM Tris, 5 mM MgCl2, 0.1 mM EDTA, pH 7.4).
  • Incubation and filtration The filtration binding assay is carried out in 96-well plates in a final volume of 250 ⁇ L per well. To each well is added 150 ⁇ L membranes (3 - 20 ⁇ g protein for cells or 50 - 120 ⁇ g protein for tissue), 50 ⁇ L of the competing test compound and 50 ⁇ L of radioligand solution in buffer. The plate is incubated at 30 °C for 60 minutes with gentle agitation. The incubation is stopped by vacuum filtration onto 0.3% PEI presoaked GF/C filters using a 96-well FilterMateTM harvester followed by four washes with ice-cold wash buffer. Filters are then dried for 30 minutes at 50 °C.
  • Saturation assays can also be used to show binding to 5-HT receptors, e.g. 5-HT1A.
  • a suitable example assay is described by Gifford Bioscience Limited (www.giffordbioscience.com - Radioligand Binding Assay Protocols). This assay is as follows: Membrane preparation: as described above for the competition assay. Incubation and filtration: The filtration binding assay is carried out in 96-well plates in a final volume of 250 ⁇ L per well.
  • membranes (3 - 20 ⁇ g protein for cells; 50 - 120 ⁇ g protein for tissue), 50 ⁇ L of the unlabeled compound (non-specifics) or buffer and 50 ⁇ L of radioligand solution in binding buffer.
  • the radioligand is added at up to 8 different concentrations (e.g. 0.2 - 20 nM).
  • the plate is incubated at 30 °C for 60 minutes with gentle agitation. The incubation is stopped by vacuum filtration onto 0.3% PEI presoaked GF/C filters using a 96-well FilterMateTM harvester followed by four washes with ice-cold wash buffer. Filters are then dried for 30 minutes at 50 °C.
  • the filter is sealed in polyethylene, scintillation cocktail (Betaplate Scint; PerkinElmer) added and the radioactivity counted in a Wallac® TriLux 1450 MicroBeta counter. Data analysis: For each radioligand concentration, non-specific binding is subtracted from total binding to give specific binding. Bound CPM values are converted to fmoles per mg protein. Data is fitted using the saturation analysis non-linear curve fitting routines in Prism® (Graphpad Software Inc). The Kd (in nM) and Bmax (fmol/mg or sites/cell) are derived from the saturation curve.
  • mice C57BL/6J male mice aged 8-12 weeks are used in the study. Water and food are provided ad libitum throughout the entire study. The holding room maintains a temperature of 21 ⁇ 1°C, humidity at 55 ⁇ 10%, and operates on a 12-hour light/dark cycle. All efforts are made to minimize animal suffering and reduce the number of animals used. Each animal are weighed at the start of the experiment.
  • Treatment Groups Mice are randomly assigned to different treatment groups, with each group receiving either a PSY series compound or a control substance (saline).
  • Test Substances Stock solutions of test substances are prepared on the same day of the experiment and are protected from direct light.
  • Administration The compounds are administered via intraperitoneal injections (i.p.), and the dosages administered are individually determined based on each animal's body weight.
  • Behavioural Assessment Immediately following the intraperitoneal injection of the drug, a mouse is placed in the open field, and video recording is initiated for 30 minutes. A camera is positioned directly above the mouse to capture its behaviour. The environment is kept quiet to minimize disturbances during the recording period.
  • Data Collection Review the recorded videos and count the number of head twitch responses displayed by each mouse during the observation period. A head twitch response is characterized by a rapid, involuntary, rotational movement of the head.
  • the aim of the present example was to screen the agonist effect of 4 test compounds on the 5- HT2A serotonin receptor activity using the U2OS 5-HTR2A Serotonin receptor Hitseeker cell line.
  • HiTSeeker 5-HTR2A/U2OS contains U2OS cells stably expressing human 5-HT2A Serotonin receptor with no tag.
  • the HiTSeeker HTR2A cell line has been designed to assay compounds or analyse their capability to modulate 5-HT2A Serotonin receptor.
  • a G protein is activated, which in turn, triggers a cellular response mediated by second messengers (Calcium). This cellular response can be measured quantifying calcium increase inside the cell determining the intensity of Fluo4.
  • Fluorescence intensity acquisition was performed in the SynergyTM 2 Multi-Detection microplate reader from Biotek. Vehicle (NaCl or H 2 O) was used as negative control; 5-HT was used as positive control. Compounds were tested at 200, 100, 50, 25, 10, 5, 2.5, 1, 0.5 ⁇ M in triplicates.
  • the U2OS 5-HTR2A Hitseeker cell line was thawed (2x10 6 cells per T25).
  • the cells were maintained in DMEM-F12 supplemented with 10% FBS at 37oC in a humidified 5% CO 2 atmosphere.
  • Day 3 The cells were plated at a concentration of 20.000 cells/well (+/-1000 cells) in 96- well plates.
  • 5-HT at 10 ⁇ M was used as positive control and vehicle (water for PSY- 1185 and PSY-1186 and NaCl for PSY-1342 and PSY-1134) as negative control.
  • Figure 15 shows the results obtained after treating the cells with the compounds. Data are presented as fluorescence intensity of Fluo-4 representing calcium increase after HTR2A stimulation. For each compound also negative control (vehicle) and 5-HT are presented. Two compounds showed an increase in intensity compared to the control: compound PSY- 1185 is the most active, exhibiting an increase of 2.76 fold compared to the control and demonstrating a clear dose-dependent activity; and compound PSY-1343 exhibit a smaller increase, 2.4 fold, and only at higher concentrations, but in a concentration-dependent manner.
  • PSY-1185 emerged as the most potent activator, showing a clear dose-response relationship across the concentrations tested, with an EC 50 value of 2.88 x 10 ⁇ 6 M. This indicates that PSY-1185 has a relatively high affinity for the 5-HT2A receptor, making it the strong agonist. PSY- 1343 exhibited measurable activity, and this was only observed at the two highest concentrations tested. These findings demonstrate the capabilities of PSY-1185 and PSY-1343.
  • SEQUENCES The sequences allocated SEQ ID Nos. herein have the following sequences: SEQ ID No: 1 Description: cDNA sequence of cytochrome P450 reductase CPR (P.

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Abstract

The present invention provides a method for producing a target alkaloid from a precursor via a metabolic pathway in a recombinant yeast cell, wherein the metabolic pathway comprises a psiH enzyme, a psiK enzyme and a psiM enzyme, the method comprising culturing the recombinant yeast cell in a culture medium comprising the precursor under conditions suitable to allow the precursor to enter the recombinant host cell such that the recombinant yeast cell produces the target alkaloid, wherein the precursor is a substrate for the psiH enzyme. Related methods and recombinant cells for use therein are also provided. The present invention further provides compounds related to psilocybin, pharmaceutical compositions comprising the same, and their use in medicine.

Description

PRODUCTION AND MEDICAL USE OF PSILOCYBIN AND RELATED COMPOUNDS TECHNICAL FIELD The present disclosure relates to novel production process for psilocybin and related compounds as well as products related to the production process, including recombinant cells in which these compounds can be produced. In addition, the present disclosure relates to novel compounds related to psilocybin, which may be produced by the production processes, and their use in medicine. BACKGROUND TO THE INVENTION Psilocybin is a naturally occurring compound of the alkaloid class, found in some species of fungi, in particular members of the genus Psilocybe, e.g. P. mexicana, P. cubensis, P. semilanceata (liberty cap toadstool) and P. cyanescens (wavy cap toadstool). Psilocybin is a prodrug; in the body psilocybin (3-[2-dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate is dephosphorylated to psilocin (4-hydroxy-N,N-dimethyltryptamine), which is a hallucinogenic compound. The hallucinogenic effects of mushrooms containing psilocybin have long been known, and psilocybin was isolated from mushrooms in the 1950s. However, due to their widespread recreational use these mushrooms as well as psilocybin and psilocin are subject to regulatory control. Nevertheless, there has been increasing interest in the use of psilocybin, psilocin, and related compounds in medicine, in particular in the field of mental health treatments. Psilocin has a similar chemical structure to serotonin (5-hydroxytryptamine), an important neurotransmitter in the human body, and is known to bind to different human serotonin receptors including the 5-hydroxytryptamine 2A (5-HT2A) receptor. Pharmaceutical drug development to date has mainly focussed on the use of psilocybin, as this is water-soluble, while psilocin is not particularly stable in any solvent. Traditionally psilocybin has been isolated from mushrooms. However, relatively recent work has led to the biosynthetic pathway of psilocybin in being discovered in 2017 by a group of scientists at the Leibniz Institute for Natural Product Research and Infection Biology (Fricke et al., September 25, 2017, Angewandte Chemie, 56(40): 12352-12355). This pathway involves four enzymes, psiD, psiH, psiK, and psiM, which produces psilocybin from the substrate 4- hydroxy-L-tryptophan. Building on this work, several publications describe the engineering of host cells to produce psilocybin using these enzymes. For example, Milne et al., (Metabolic Engineering, 60 (2020): 25-36) report the metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives beginning with the native production by the S. cerevisiae cells of tryptophan. Rather than use psiD at the start of the psilocybin biosynthetic pathway, the authors reported the use of the Catharanthus roseus tryptophan decarboxylase (due to its confirmed efficiency in S. cerevisiae) to convert the tryptophan into tryptamine, which is then acted on by the psiH enzyme, with the psiK and psiM enzymes being used in subsequent steps. The authors report that using their method resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. However, there remains the need to improve biotechnological production of psilocybin, psilocin, and related compounds, in order to meet the ongoing medical need for psilocybin and psilocin, as further clinical trials and research are conducted on these compounds. There also remains a need for the identification of compounds related to psilocybin, in order to broaden the range of medicinal compounds available for use in medical treatment, and in particular in the field of mental health treatments. SUMMARY OF THE INVENTION In a first aspect the present invention provides a method for producing a target alkaloid from a precursor via a metabolic pathway in a recombinant yeast cell, wherein the metabolic pathway comprises a psiH enzyme, a psiK enzyme and a psiM enzyme, the method comprising culturing the recombinant yeast cell in a culture medium comprising the precursor under conditions suitable to allow the precursor to enter the recombinant yeast cell such that the recombinant yeast cell produces the target alkaloid, wherein the precursor is a substrate for the psiH enzyme. The present invention further provides a recombinant yeast cell comprising a psiH gene, a psiK gene, and a psiM gene, wherein the recombinant yeast cell: (i) does not comprise an L- tryptophan decarboxylase gene; and/or (ii) comprises an exogenous CPR gene and an adenosylhomocysteinase (SAH1) gene. In addition, the present invention provides a yeast cell culture produced by the method of the invention described above and comprising the recombinant yeast cell and the target alkaloid. Further provided is a medical formulation comprising the recombinant yeast cells containing the target alkaloid and at least one excipient. In addition, the present invention provides a method for extracting a produced target alkaloid comprising an amine group and an -HPO4 group from a recombinant yeast cell, the method comprising: (i) contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell; (ii) centrifuging the extraction mixture to form a supernatant comprising the target alkaloid; (iii) ensuring the supernatant has a pH between 10 and 14 and has a low ionic strength solution such that the charge of N in the amine group is neutral; (iv) applying the supernatant to an anion exchange column; and (v) using a hydroxyl ion gradient to elute at least one fraction comprising the target alkaloid. In one embodiment the amine group is selected from -N(CH3)2 and an -NHCH3 group. In addition, a yeast cell extract prepared by this method is also provided. In a further aspect, the present invention provides a method for increasing methylation capacity of a psiM enzyme in a recombinant yeast cell, the method comprising increasing expression of an adenosylhomocysteinase (SAH1) enzyme in the recombinant yeast cell so as to increase the capacity of the recombinant yeast cell to hydrolyse S-adenosyl-l-homocysteine. In addition, the present invention provides a compound according to Formula (I), or a pharmaceutically acceptable salt thereof, wherein: X is S, O, C, or N, wherein when X is S or O, R8a and R8b are absent, and wherein when X is N, R8b is absent, Z is C or N, wherein when Z is N, R2 is absent, and wherein when X is N, Z is N, R4 is -HPO4, or -OH, Y1 is -N+H(CH3)2, or -N+H2 CH3, R5, R6, R7 are independently selected from H, D, -CFH2, -CHF2 or -CF3, R2, R3a, R3b, R3c, R3d, R8a and R8b are independently selected from H, or D. In addition, the present invention provides a compound according to Formula (IA):
Figure imgf000006_0001
wherein: R4 is -HPO4, or -OH, Y1 is -N+H(CH3)2, or -N+H2 CH3, R5, R6, R7 are independently selected from H, D, -CFH2, -CHF2 or -CF3, R2, R3a, R3b, R3c, R3d are independently selected from H, or D. Preferably in formula IA, R2, R3a, R3b, R3c, R3d, R5, R6, R7 are all H. In addition, the present invention provides a compound according to Formula (IB):
Figure imgf000007_0001
wherein: R4 is -HPO4, or -OH, Y1 is -N+H(CH3)2, or -N+H2 CH3, R5, R6, R7 are independently selected from H, D, -CFH2, -CHF2 or -CF3, R2, R3a, R3b, R3c, R3d are independently selected from H, or D. Preferably in formula IA, R2, R3a, R3b, R3c, R3d, R5, R6, R7 are all H. Where the present disclosure mentions Formula I and features of Formula I, Formula IA and IB are also intended, unless other details of the specific disclosure dictate otherwise. In particular, the target alkaloids according to formula IA and IB may also be produced by the methods of the invention and may be used in the products and compositions of the invention. The present invention further provides a pharmaceutical composition comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof. Still further, the present invention provides the compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in medicine. The present invention provides improved methodology for the production of target alkaloids, and especially psilocybin, psilocin and related compounds in recombinant yeast cells. In particular, the method described herein advantageously avoids the need to rely on endogenously generated L-tryptophan as the first substrate for the metabolic pathway and instead begins with a later substrate (also referred to herein as precursor) that is the substrate for the psiH enzyme. The present inventors have surprisingly shown that suitable conditions can be obtained that allow this later substrate in the pathway to be efficiently taken up by the cells. As a result, the methods described herein can be used to obtain superior conversion rates and titres of the target alkaloid. In addition, the present invention provides purification methods that advantageously lead to the efficient and effective purification of the target alkaloids. In particular, the purification methods can be used to avoid the need for cell disruption and chromatographic separation steps. Still further, the present invention provides novel compounds, pharmaceutical compositions comprising these compounds, and their use in medicine. BRIEF DESCRIPTION OF THE DRAWINGS To assist understanding of the present disclosure and to show how embodiments may be put into effect, reference is made by way of example to the accompanying drawings in which: Figure 1 provides a schematic of one example of the biosynthetic route of psilocybin production according to present invention. Figure 2 is a schematic showing the vector map for pPpGAP-KanS-HIS4-psiM (P. cyan.)-psiK (P. cub.)-psiH (P. cyan.)-CPR (P. cyan.)-SAH1 (P. p.). Figure 3 is a picture showing SDS-PAGE analysis of selected P.pastoris strains having psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus and an additional copy of endogenous P. pastoris SAH1 (Adenosylhomocysteinase) gene integrated into the genome. Figure 4 provides results of HPLC analysis of an extract of yeast cells converting tryptamine to psilocybin. Figure 5 provides a chromatogram of the first step of psilocybin purification using 150 ml volume (XK 26/40) SepharoseQ HP column. Figure 6 provides a chromatogram of the second step of psilocybin purification using 150 ml volume (XK 26/40) SepharoseQ HP column. Figure 7 provides HPLC analysis of psilocybin product at 230 nm of combined 22-32 fractions after second step purification using Sepharose HP. Figures 8A and 8B provide analysis showing production of Substance (4) from Example 2. Figure 8A provides a chromatogram from HPLC analysis showing production of Substance (4). Figure 8B provides a mass spectrum for Substance (4). Figures 9A and 9B provide analysis showing production of Substance (5) from Example 2. Figure 9A provides a chromatogram from HPLC analysis showing production of Substance (5). Figure 8B provides a mass spectrum for Substance (5). Figure 10 provides a chromatogram of Substance (5) from Example 2 purification, using 150 ml volume (XK 26/40) SepharoseQ HP. Figures 11A-C provide chromatograms from HPLC analysis of whole cell broth (WCB) extract after addition of 2mM substrate – 2- (Benzofuran-3-yl) ethylamine (Substance (1) Example 3). Figure 11A shows result 5 mins after addition. Figure 11B shows result 4 hours after addition. Figure 11C shows result 20 hours after addition. Figure 12 provides a chromatogram of the first step of O-Psilocybin purification (in Example 3) using a 150 ml volume (XK 26/40) SepharoseQ HP column. Figure 13 provides a chromatogram of the second step of O-Psilocybin purification (in Example 3) using a 150 ml volume (XK 26/40) SepharoseQ HP column. Figure 14 provides HPLC analysis of combined 11-13 fractions after the second step purification of O-Psilocybin (in Example 3) using Sepharose HP. Figure 15 provides bar charts showing the dose-response for compounds tested in the 5-HT2A serotonin receptor agonist assay. Cells were treated with the test compounds at serial concentrations. Data points represent the mean ± SD for each condition for a single experiment performed in triplicate. Results are expressed as the fluorescence intensity (arbitrary units, a.u.) of Fluo-4. C- represents vehicle. Figure 16 provides the dose response curve for PSY-1185 tested in the 5-HT2A serotonin receptor agonist assay. Data is presented as the percentage of activity of the HTR2A normalized against the zero control (vehicle) that represents the 0% of activity and the maximum concentration of each compound that represents the 100%. DETAILED DESCRIPTION As noted above, the biosynthetic pathway for the production of psilocybin was discovered in 2017 by a group of scientists at the Leibniz Institute for Natural Product Research and Infection Biology (Fricke et al., September 25, 2017, Angewandte Chemie, 56(40): 12352-12355). This route (which is shown in Figure 1) uses the enzymes psiD, psiH, psiK, and psiM, to produce psilocybin from the precursor tryptophan; tryptophan being produced in the cell via other metabolic pathways, e.g. the Shikimate-Chorismate pathway as described in Milne et al., (Metabolic Engineering, 60 (2020): 25-36). Further work in this area has been done by Vogan et al., (US 11,441164 B2, published 13 September 2022), which suggested the construction of S. cerevisiae yeast strains that have been genetically engineered to increase the metabolic flux towards L-tryptophan. Also suggested by Vogan et al., is feeding L-tryptophan to the recombinant host organism by media supplementation and take up by host cells expressing the recombinant TAT2 importer protein. Nevertheless, in these methods the obtained levels of produced psilocybin remain relatively low relatively low or lack experimental demonstration. As described above, the present inventors have identified a new method using the psiH, psiK and psiM enzymes for producing a target alkaloid (including psilocybin) from a precursor via a metabolic pathway in a recombinant yeast cell. In particular, the present inventors have found that it is possible to improve the production of the target alkaloid by starting the metabolic pathway with a precursor that is a substrate for the psiH enzyme (as shown in Figure 1), since suitable conditions can be created in the culture media that allow the precursor to be efficiently taken up by the yeast cells. This route is advantageous as it is not limited to cell-produced tryptophan. The target alkaloid may be according to formula (I): in which X is N, S, O or C, wherein when X is N, R8b is absent, and wherein when X is S or O, R8a and R8b are absent, Z is C or N, wherein when Z is N, R2 is absent, R4 is -HPO4, or OH, Y1 is -N+H(CH3)2, or -N+H2 CH3, R5, R6, R7 are independently selected from H, D, -CFH2, -CHF2 or -CF3, R2, R3a, R3b, R3c, R3d, R8a and R8b are independently selected from H or D. The precursor can be selected based on the target alkaloid and the reactions that the enzymes psiH, psiK and psiM catalyse in the metabolic pathway. While these enzymes have been shown to produce psilocybin (as shown in one example of the invention in Figure 1), the examples provided herein surprisingly show that the enzymes are able to accommodate variations in their substrates (in particular in locations on the substrate molecules that are away from positions involved in the enzymatic reaction), allowing the method described herein to be used to produce compounds that are related to psilocybin. In one example of the invention, X is N and Z is C, and more preferably the target alkaloid is psilocybin. Where the target alkaloid is psilocybin the precursor can be tryptamine. In further examples of the invention, where X is S, O or C, Z is C, and wherein R2, R3a, R3b, R3c, R3d, R5, R6, and R7, are all H, the precursors can be 2-(benzothien-3-yl)ethylamine, 2- (Benzofuran-3-yl)ethylamine and 2-(1H-inden-3-yl)ethanamine, respectively. In particular, the experimental work described herein shows that the alteration of the substrates of psiH, psiK and psiM enzymes does not prevent these from catalysing the reactions. In further examples of the invention, X is N, Z is N and R2 is absent, and R3a, R3b, R3c, R3d, R5, R6, R7,R8a and R8b are all H. In this embodiment the precursor can be 2-(1H-indazol-3- yl)ethanamine hydrochloride. Preferably in R5, R6, R7 are independently selected from H, and D, and are more preferably H. Preferably Y1 is -N+H(CH3)2. The enzymes that may be used in the method of the present invention are as follows: PsiH Enzyme - The psiH enzyme is a tryptamine 4-monooxygenase, which is able to catalyse the conversion of tryptamine to 4-hydroxytryptamine. The psiH enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens. The psiH enzyme may be produced from a cDNA sequence having SEQ ID NO: 7. In a preferred embodiment the psiH enzyme comprises an amino acid sequence having SEQ ID NO: 8, or an amino acid sequence having at least 85% identity to SEQ ID NO: 8. More preferably the psiH enzyme comprises an amino acid sequence having SEQ ID NO: 8, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 8. PsiK Enzyme – The psiK enzyme is a 4-hydroxytrptamine kinase, which is able to catalyse the conversion of 4-hydroxytryptamine to norbaeocystin. The psiK enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens. The psiK enzyme may be produced from a cDNA sequence having SEQ ID NO: 3. In a preferred embodiment the psiK enzyme comprises an amino acid sequence having SEQ ID NO: 4, or an amino acid sequence having at least 85% identity to SEQ ID NO: 4. More preferably the psiK enzyme comprises an amino acid sequence having SEQ ID NO: 4, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 4. PsiM enzyme – The psiM enzyme is a methyltransferase, which is able to catalyse the methylation of norbaeocystin to form baeocystin, and the subsequent methylation of baeocystin to form psilocybin. The psiM enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens. The psiM enzyme may be produced from a cDNA sequence having SEQ ID NO: 5. In a preferred embodiment the psiM enzyme comprises an amino acid sequence having SEQ ID NO: 6, or an amino acid sequence having at least 85% identity to SEQ ID NO: 6. More preferably the psiM enzyme comprises an amino acid sequence having SEQ ID NO: 6, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 6. Cytochrome P450 reductase (CPR) – a CPR enzyme may also be used in the method of the invention. In particular, as reported previously (e.g. WO 2021/052989 A1), yeast endogenous P450 reductases are not compatible with psiH. Accordingly, it is preferred to use a recombinant yeast cell expressing an exogenous CPR gene in order to increase psiH efficiency. The CPR enzyme may be derived from Psilocybe cyanesens, and may be produced from a nucleotide sequence having SEQ ID NO: 1. In a preferred embodiment the CPR enzyme comprises an amino acid sequence having SEQ ID NO: 2, or an amino acid sequence having at least 85% identity to SEQ ID NO: 2. More preferably the CPR enzyme comprises an amino acid sequence having SEQ ID NO: 2, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 2. Adenosylhomocysteinase (SAH1) – the SAH1 enzyme may also be used in the method of the invention to improve the efficiency of the psiM methylation steps. In particular, the present inventors have surprisingly found that the S-adenosyl-L-homocysteine (SAH), the molecule that is formed when a methyl group is transferred from SAM to a target molecule, acts as competitive inhibitor of the SAM-dependent methyl transferase reactions catalysed by psiM. The yeast cell may already comprise an endogenous SAH1 gene. However, one or more (additional) copies of a SAH1 gene may be incorporated into the recombinant yeast cell. The gene may be derived from P. pastoris, and may comprise the nucleotide sequence of SEQ ID NO: 9. In a preferred embodiment the SAH1 enzyme comprises an amino acid sequence having SEQ ID NO: 10, or an amino acid sequence having at least 85% identity to SEQ ID NO: 10. More preferably the SAH1 enzyme comprises an amino acid sequence having SEQ ID NO: 10, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: In contrast to the methods of the prior art, the recombinant yeast cell used in the method of the invention does not comprise a psiD gene or other gene encoding a tryptophan decarboxylase. Further, in view of the finding regarding the utility of SAH1 to improve the efficiency of the methylation step involving psiM the present invention, a further aspect of the invention relates to a method for increasing methylation capacity of a psiM enzyme in a recombinant yeast cell. The method comprises increasing expression of an adenosylhomocysteinase (SAH1) enzyme in the recombinant yeast cell so as to increase the capacity of the recombinant yeast cell to hydrolyse S-adenosyl-l-homocysteine. In particular, expression may be increased by transforming the yeast cell with one or more copies of an SAH1 gene encoding the SAH1 enzyme; and/or increasing transcription from an SAH1 gene encoding the SAH1 enzyme using a promoter or an enhancer. Such a method may be used as part of a method for producing a target alkaloid from a precursor as described above. Alternatively, it may be used in combination with the psilocybin production methods already described in the prior art. As shown in Figure 1, in an example of the method of the present invention the psiM enzyme carries out two methylation reactions to produce a target alkaloid in which Y1 is -N+H(CH3)2, however, by adding more substrate and/or by restricting the methylation capacity of the psiM enzyme a target alkaloid in which Y1 is -N+H2 CH3 can be produced. The present disclosure provides recombinant yeast cells that can be used in the methods described herein to produce the target alkaloid. Types of yeast cell that are suitable for performing the method of the invention are those already know in the art for recombinant protein production. For example, the yeast may be from the genus Pichia, such as Pichia pastoris, from the genus Sacchromyces, such as S. cerevisiae or S. pombe, or from the genus Yarrowia such as Y. lipolytica. Preferably the recombinant yeast cell is from the genus Pichia, and most preferably the yeast cell is Pichia pastoris. Recombinant vectors carrying the genes for the enzymes described above can be transformed into the yeast cell by methods known in the art. It is preferred that the DNA constructs are integrated into the yeast genome in order to provide stable expression of the genes. The genes for the enzymes described above can be placed under the control of inducible or constitutive promoters or gene expression control elements. Suitable promoters are known in the art for the production of recombinant proteins in yeast. However, where the yeast is Pichia, it is preferred that the promoters AOX1 or GAP are used. The GAP promoter is most preferred. As noted above, in the method of the present invention the recombinant yeast cell is cultured in a culture medium comprising the precursor under conditions that are suitable to allow the precursor for the enzyme psiH to enter the recombinant host cells. In particular, the precursors for the enzyme psiH are charged molecules in normal yeast growth conditions, which do not effectively cross the yeast cell membrane. However, the present inventors have surprisingly found that they are able to culture the recombinant yeast cells at a pH of 6 to 7.8, preferably at a pH of 7.5 to 7.8 and most preferably at a pH of 7.8. Although these pH values are not particularly close to the pKa of the precursor molecules (with the pKa of tryptamine being approximately 10.2 at which yeast cannot be readily cultivated), the present inventors have observed rapid diffusion of the precursor into the yeast cell, as a first step to efficient production of the target alkaloid. At such pH values it is important to use a phosphate source that does not precipitate. A suitable phosphate source in the culture media is therefore sodium hexametaphosphate. The precursor may be added to the culture media in batches, and one batch may be added after the full conversion of the previously added precursor has been achieved. In particular, it has been found that the constant addition of precursor may lead to an accumulation of intermediates. In a preferred example, the precursor is added to the culture media at a rate of 4 to 6 μM every 2.5 to 3.5 hours per 1 gram wet cell weight. As shown by the Examples provided herein the method according to the present invention can achieve advantageous production rates of the target alkaloid. Specifically, the target alkaloid can be produced at a rate of at least 1 g/litre of culture every 24 hours, preferably at least 3 g/litre of culture every 24 hours, more preferably at least 5g/litre of culture every 24 hours. In a further aspect the present invention provides a method for extracting a produced target alkaloid comprising an amine group and an -HPO4 group from a recombinant yeast cell, the method comprising: (i) contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell; (ii) centrifuging the extraction mixture to form a supernatant comprising the target alkaloid; (iii) ensuring the supernatant has a pH between 10 and 14 and has a low ionic strength solution such that the charge of N in the -N(CH3)2 group is neutral; (iv) applying the supernatant to an anion exchange column; and (v) using a hydroxyl ion gradient to elute at least one fraction comprising the target alkaloid. In particular, the amine group may be an -N(CH3)2 group or an -NHCH3 group. In (i) of the above method, the whole cell broth (comprising the recombinant yeast cell) may be contacted with the acetonitrile to form an extraction mixture. In such cases, step (iii) would normally include a step of buffer exchange, e.g. via nanofiltration, to transfer the target alkaloid into a suitable buffer so that it can bind to the anion exchange column. Alternatively, prior to (i) the whole cell broth may be centrifuged to separate the yeast cells from the growth media and the yeast cells washed in order to remove the media and placed the cells in a suitable media prior to contact with the acetonitrile. The solution for extraction in (i) may comprise 15 to 75% (v/v) acetonitrile, preferably 20 to 60% (v/v) acetonitrile, more preferably 25 to 35% (v/v) acetonitrile. In (iii) it is ensured that the supernatant has a pH between 10 and 14 and a low ionic strength such that the charge of the N in the -N(CH3)2 group is neutral. Such low ionic strength solution is a solution containing 0.05mM to 50 mM ions wherein the ions have a lower selectivity towards the positively charged groups of the anion exchange column than the -HPO4 group. In this way it can be ensured that the phosphate groups of the target alkaloid bind to the column. An example of a suitable low ion strength solution is 0.5 mM to 1.5 mM NaOH. Other low ionic strength solutions can be readily envisaged by a person skilled in the art. The anion exchange column may be a Sepharose column, e.g. Sepharose Q HP, Sepharose Q, or Sepharose DEAE. In step (v) a hydroxyl ion gradient is used to elute at least one fraction comprising the target alkaloid. In particular, a hydroxyl ion gradient has been found by the inventors to produce the best selectivity for the target alkaloid. The at least one fraction may be two or more fractions and the method may comprise combining the two or more fractions, diluting the combined fractions and reapplying to the anion exchange column, and repeating step (v). In a further aspect the present invention provides novel compounds related to psilocybin and to psilocin. These compounds can be prepared by the methods described above. A compound according to this invention is according to Formula (I), or a pharmaceutically acceptable salt thereof,
Figure imgf000017_0001
wherein: X is S, O, C, or N, wherein when X is S or O, R8a and R8b are absent, and wherein when X is N, R8b is absent, Z is C or N, wherein when Z is N, R2 is absent, and wherein when X is N, Z is N, R4 is HPO4, or OH, Y1 is -N+H(CH3)2, or -N+H2 CH3, R5, R6, R7 are independently selected from H, D, -CFH2, -CHF2 or -CF3, R2, R3a, R3b, R3c, R3d, R8a and R8b are independently selected from H, or D. In some examples, X is S or O, R8a and R8b are absent, Z is C, and R2 is H. In some examples, X is C, Z is C, and R8a, R8b, and R2 are H. In some examples, X is N and Z is N, R8b and R2 are absent, and R8a is H. Preferably in these examples Y1 is -N+H(CH3)2. Preferably in these examples R5, R6, R7 are independently selected from H and D, and are more preferably H. Preferably in these examples, R3a, R3b, R3c, R3d, R5, R6, and R7 are all H. The above compounds may be comprised in a pharmaceutical composition. In particular, the pharmaceutical composition may comprise the compound and a pharmaceutically acceptable excipient, such as a pharmaceutically acceptable carrier. The compound may be comprised in the pharmaceutical composition in a therapeutically effective amount. The pharmaceutically acceptable salt of the compound may be HCl, HI, HBr, HF, ascorbate, or hydrofumarate, 5-fumarate, oxalate, or maleate. The compounds, and pharmaceutical compositions comprising the compounds, are for use in medical therapy by administration to a subject in need thereof and may be used for the treatment and/or prevention of a disease or a disorder in a subject. The subject may be a non-human mammal or a human. Preferably the subject is a human subject. Alternatively, the present invention provides methods of prevention and treatment using the compositions and compounds described herein, the methods comprising administering an effective amount of the composition or compound to a subject in need thereof, wherein the subject is preferably a human subject. The methods can be used to achieve the therapeutic effects and/or the prevention and treatment of the diseases/disorders mentioned below. The compounds or pharmaceutical compositions may be for administration by any appropriate route. Preferably the compounds or pharmaceutical compositions are for oral, subcutaneous or intravenous administration. More preferably the compounds or pharmaceutical compositions are for oral administration. The compound according to formula I above may act as a prodrug, i.e., it may be metabolized in the body of the subject to produce a drug which has the medicinal effect. For example, in the same manner that psilocybin is known to be metabolized to psilocin once ingested, a compound of formula I having an -HPO4 group at position R4 may also lose the phosphate group once ingested, and be converted to the active drug having an -OH group at position R4. The compounds, and pharmaceutical compositions comprising the compounds, may be used in medical therapy as an agonist of a serotonin receptor, or as a modulator of a serotonin receptor. For example, the compound (and pharmaceutical composition comprising the compounds) may be for use as an agonist of a 5-hydroxytryptamine (5-HT) receptor, such as 5-HT2A receptors or 5-HT1A receptors. In particular, the following is known about the main mechanisms of action of psilocybin, to which the present compounds are related, in relation to the treatment of mental health conditions/disorders: Serotonin Receptor Modulation: Psilocybin (and most classic psychedelics of tryptamine and ergotamine classes) primary mechanism of action is through the modulation of serotonin receptors in the brain. Serotonin is a neurotransmitter that plays a crucial role in mood regulation and various mental health conditions. Psilocybin binds to serotonin receptors, particularly the 5-HT2A receptor, which may lead to changes in neuronal connectivity and function. These changes might impact the neural circuits associated with these conditions. Default Mode Network (DMN) Disruption: The mental health conditions/disorders listed below are associated with abnormal activity in the default mode network, a network of brain regions involved in self-referential thoughts and rumination. Psilocybin has been shown to disrupt the DMN, leading to a temporary alteration in the perception of self and ego dissolution. This disruption might offer relief from the rigid thought patterns and compulsions characteristic of depression, OCD, etc. Neuroplasticity: Psilocybin has been proposed to promote neuroplasticity, the brain's ability to reorganize itself by forming new neural connections. In the context of OCD, promoting neuroplasticity may help individuals break free from obsessive thought patterns and compulsive behaviors by allowing the brain to rewire itself in a more adaptive way. Psychological Insights: Psychedelic experiences induced by psilocybin often involve intense introspection and the surfacing of repressed emotions or memories. This can lead to psychological insights and catharsis, which may be particularly relevant in the treatment of depression, where deep-seated fears and anxieties often drive compulsive behaviors. (References: Andersen et al., Acta Psychiatr. Scand., 2021, Feb; 143(2):101-118; Strumila & Nobile et al., Pharmaceuticals, 2021, 14, 1213). Accordingly, in one embodiment the compound or pharmaceutical composition is for use in treating or preventing a mental health condition or disorder, such as one selected from anxiety disorder, bipolar disorder, dementia, ADHD, schizophrenia, OCD, autism, PTSD, addiction/substance abuse, post-partum depression, suicidal thoughts, and depression. Preferably the mental health condition or disorder is treatment-resistant depression or major depressive disorder. In another embodiment the compound or pharmaceutical composition is for use in treating a neurodegenerative disease or disorder, or a brain injury, in particular by increasing neuroplasticity. The neurodegenerative disease may be Alzheimer’s disease or Parkinson’s disease. The brain injury may be caused by a stroke. In particular, the following is known about the main mechanisms of action of psilocybin, to which the present compounds are related, in relation to the treatment of neurodegenerative diseases or disorders: Neuroplasticity: psilocybin induces synaptic, structural, and functional changes, particularly in pyramidal neurons in the prefrontal cortex. These encompass heightened glutamate exocytosis, activation of α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), the involvement of brain- derived neurotrophic factor (BDNF) and signalling pathways mediated by mammalian target of rapamycin (mTOR), the upregulation of synaptic protein expression, and the promotion of synaptogenesis. Antioxidant effects: It is known that indole ring-containing molecules, including psilocybin, exert antioxidant effects. (References: Moliner et al., Nature Neuroscience, Vol. 26, June 2023: 1032-1041; Kozlowska et al., Journal of Neurochemistry, 2022; 162:89-108; Strumila & Nobile et al., Pharmaceuticals, 2021, 14, 1213.) In a further embodiment the compound or pharmaceutical composition is for use in treating or preventing an estrogen-related disease. In particular, some benzothiophene-class compounds are known to act as selective estrogen receptor modulators (SERMs), and are used to treat osteoporosis, and some breast cancers in postmenopausal women. Moreover, some benzothiophene class compounds have been show to exert antiproliferative (IC50) effects in various breast cancer type cell lines (Bai et al., European Journal of Medicinal Chemistry, 221 (2021): 113543). Accordingly, the compounds / pharmaceutical compositions described herein may be for use in treating an estrogen-related disease, such as cancer (estrogen receptor positive breast cancer), infertility caused by ovulatory dysfunction, and post-menopause osteoporosis. In particular, the compound may be one which X in formula I is S. In a still further embodiment the compound or pharmaceutical composition may be for use in treating or preventing an autoimmune disease, such as rheumatoid arthritis, multiple sclerosis, Crohn’s disease, psoriasis, psoriatic arthritis, or type 1 diabetes. In particular, the compound or pharmaceutical may be for use as an anti-inflammatory agent, or for use in the treatment or prevention of inflammation, preferably in the treatment of autoimmune disorders, including those indicated above. In this regard, 5-HT2A receptor agonists have been shown to have anti-inflammatory effects, for example by Flanagan and Nichols, 2018 (“Psychedelics as anti-inflammatory agents”, Int. Rev. Psychiatry, 2018), potentially by their immunomodulatory activity and in part through the mobilization of cell- intrinsic neuroprotective mechanisms. It should be noted that there is a higher risk of developing clinical depression or mood disorders if one has been diagnosed with an autoimmune condition (Siegmann et al., JAMA Psychiatry. 2018 Jun 1;75(6):577-584). It is suggested that the symptoms of depression and anxiety could potentially emerge because of autoimmune processes and the inflammation occurring within the nervous system. This may occur either through the dysregulated inflammatory cytokines, establishing a feedback loop between immune cells residing in the periphery and those in the brain, or via inflammation originating within the nervous system itself. In particular, as described by Thompson & Szabo, 2020 (Immunology Letters, 228 (2020) 45-54): “Functional studies showed that 5-HT modulates the release of IL- 1beta, IL-6, IL-8/CXCL8, IL-12p40 and TNF-α, (…). 5-HT can also modulate human macrophage polarization and dendritic cell functions, and can contribute to the maintenance of an anti-inflammatory state via 5-HT2B and 5-HT7 receptor binding [67,68]. Given the role of the 5-HT system in immune-modulation and inflammatory properties, it is highly likely that there are undiscovered immune and inflammatory effects from exposure to psychedelic compounds due to their serotonergic activity.(…). The Sig1R plays a fundamental role in the regulation of different cellular mechanisms such as mitochondrial function, apoptosis, proliferation, and neuroprotection [69,70]. Sig1R also modulates inflammatory and immune responses by regulating the activation of the transcription factors nuclear factor kappa B (NF-кB) and several mitogen-activated protein kinases (MAPKs) [71]. Both NF-кB and MAPKs are important regulators of gene transcription involving immune responses and the production of inflammatory cytokines. Abnormal Sig1R functions have been implicated in a number of psychiatric and inflammatory-related conditions such as MDD, Alzheimer’s disease, Parkinson’s disease, cardiovascular disease, immune reactions, and proliferation of cancer cells.”. Accordingly, the compounds described herein can be used in the treatment and/or prevention of disease. In addition, the present invention also provides compositions/formulations which are related to their method of production in yeast. In particular, the present invention further provides a yeast cell culture produced by the method described herein comprising the recombinant yeast cell of and the target alkaloid. These cell cultures may be used to store the target alkaloid, e.g. for the purposes of transportation. For example, they may further comprise a storage buffer such as one that is suitable to stabilize the cells. The yeast cell culture produced by the method described herein may also be processed to form a medicinal formulation comprising the recombinant yeast cells containing the target alkaloid. These formulations can be produced by centrifuging the yeast cell culture produced by the methods described herein to remove the cell broth, and formulating the cells in a solution or carrier suitable for human consumption. The present invention also provides a yeast cell extract produced by contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell, and centrifuging the extraction mixture to form the yeast cell extract that is the supernatant comprising the target alkaloid. Such extracts may also be used to store / transport the target alkaloid. The invention is demonstrated by the following examples. These are intended as examples only and do not limit the present disclosure. EXAMPLES Example 1 - Highly efficient conversion of Tryptamine to Psilocybin in the yeast P. pastoris cells using enzymes from fungi of Psilocybe genus and optimized yeast SAM (S- Adenosylmethionine) cycle In this example psilocybin is synthesized from tryptamine in 4 reactions: 1. Tryptamine to 4-hydroxytryptamine (hydroxylation of tryptamine at fourth position of indole) catalyzed by psiH enzyme (with CPR (cytochrome P450 reductase) to improve efficiency of psiH function, because yeast endogenous P450 reductases are not compatible with psiH). 2. 4-hydroxytryptamine to norbaeocystin (phosphorylation of hydroxy group at fourth position of indole) catalyzed by psiK. ATP is required for this reaction. 3. Norbaeocystin to baeocystin (1st methylation of 2-aminoethyl group) catalyzed by psiM. SAM is required for this reaction. 4. Baeocystin to psilocybin (2nd methylation of 2-aminoethyl group) catalyzed by psiM. SAM is required for this reaction. Yeast Strain and Promoter A well-studied Pichia pastoris strain deposited as CBS7435 (CBS, Centraalbureau voor Schimmelcultures) was selected. (Strain as described e.g. by Küberl et al., “High-quality genome sequence of Pichia pastoris CDS7435”, Journal of Biotechnology; 154(4); 20 July 2011: pages 312-320.) The wild-type Pichia genome has two alcohol oxidase genes (AOX1 and AOX2), and in the presence of methanol the enzymes produced from these genes, AOX, allow the yeast to be grown on methanol. The AOX1 promoter (pAOX1) is tightly regulated and strongly inducible by methanol. Knocking out the AOX1 gene leads to methanol utilization slow phenotype, “MutS”. However, in a “Mut+ strain” yeast both AOX1 and AOX2 are present and functional. The glyceraldehyde-3-phosphate dehdrogenase promoter (pGAP) is known as a strong constitutive promoter in yeast. Several different P. pastoris strains were prepared with vectors carrying the psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus integrated into the yeast genome, having different Tryptamine to psilocybin conversion properties. Genes were placed under strong constitutive GAP promoter (enzymes are produced constantly) or very strong inducible AOX1 promoter (enzymes are produced only when methanol is added). The following strains were created: CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1; CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_2, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_5, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_8, CBS7435_pPpAOX1-HIS4-psiM-psiK-psiH-CPR_1_MutS, CBS7435_pPpAOX1-HIS4-psiM-psiK-psiH-CPR_2_Mut+. The HIS4 gene (encoding histidinol dehydrogenase – EC 1.1.1.23) is included in the vector for selection of transformants on histidine-deficient medium. Our results (not presented here) show that psilocybin production is better in P. pastoris strains where the target genes are placed under the pGAP promoter. In particular, we note that the pGAP promoter provides the following advantages: (a) the amount of enzymes is optimum for conversion rate/speed of tryptamine to psilocybin without depleting cellular metabolites necessary for enzymatic reactions; (b) there is no need to use methanol, which is toxic, and requires safety protocols and equipment, and therefore avoiding its use makes the technology greener; and (c) use of pGAP avoids the oxidative stress in the cells caused by methanol metabolism, helping to avoid the formation of psilocin polymers. Fermentation conditions P. pastoris fermentation conditions, that allow uptake of tryptamine to the cell, were developed. Under normal yeast fermentation conditions, pH 5.0, tryptamine is a charged molecule that cannot cross yeast cells plasma membrane. Thus, we developed a fermentation media, that allows yeast cell growth under pH 7 – 7.8. Under such pH tryptamine is partly uncharged and can cross plasma membrane. Most of the phosphate salts used for yeast fermentation precipitate under pH 7 – 7.8. Instead of common phosphate salts or phosphoric acid we use sodium hexametaphosphate (HMP) as phosphate source. It allows to grow P. pastoris in wide range of pH (3 – 7.8). Also, pH 7-7.8 of the media avoids dephosphorylation of psilocybin to psilocin, because either it is retained in the cell, or if it is secreted into the culture media, the basic pH is favorable for the stability of the molecule. (Chemically defined media, compliant with GMP requirements, can be used for fermentation of P. pastoris.) SAM cycle improvement Strains created: CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAM2_1, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAM2_2, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAM2_6, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_5, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_8, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAM2-SAH1_5, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAM2-SAH1_7. Using the CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1 strain mentioned in the section above, we have found that the rate of conversion of tryptamine to psilocybin rate is 5 µM of tryptamine to psilocybin in 3-6 hours per 1 gram of wet cell weight (WCW). The addition of tryptamine is done in batches after full conversion of previously added tryptamine is achieved. Constant addition of tryptamine exhausts cellular SAM, as evident by accumulation of baeocystin and, especially, norbaeosystin, and is detrimental for the cells. The average amount of WCW during conversion is about 200 g/L, so the conversion rate is 1 mM of tryptamine to 1 mM psilocybin per 3–6 hours (285 mg of psilocybin is synthesized in 3-6 hours per 1L of medium). Our research has shown that this conversion rate is limited by the availability of SAM in yeast cells. The hydroxylation and phosphorylation reactions, i.e. conversion of tryptamine to norbaeocystin, are not limited and are carried out by psiH and psiK enzymes almost instantly. On the other hand, the double methylation reaction (carried out by psiM) is limited by the availability of SAM to such an extent that addition of 10-15 µM of tryptamine per 1 g of WCW is detrimental to the cells. The amount of produced SAM in the cell is also responsible for slowing down the conversion of 5 µM of tryptamine per 1 g of WCW to psilocybin from 3 hours after first addition of tryptamine to 6 hours after third and further addition of tryptamine. Using such setup, about 1.5 – 2 g of psilocybin per 1 L of yeast fermentation media can be produced in 24 hours. Most of it remains in yeast cells and about 10-15 % is found in the yeast growth media. Accordingly, yeast P. pastoris strains with optimized SAM cycle and increased rate of psilocybin synthesis were generated. SAM is synthesized by SAM synthase by adenosylation of methionine. Our research showed that the addition of methionine did not increase the rate of norbaeocystin methylation, thus there is a lack of SAM synthesis capabilities in yeast cell. However, integration of an additional copy of S. cerevisiae SAM2 (S-adenosylmethionine synthase 2) gene only increased the rate of norbaeocystin methylation marginally (for this experiment a yeast P. pastoris strain that had psiH, psiK, psiM, CPR genes from Psilocybe genus and SAM2 from S. cerevisae integrated into the genome was generated. All 5 genes were placed under a GAP promoter. We postulate that S-adenosyl-L-homocysteine (SAH), the molecule that is formed when a methyl group is transferred from SAM to a target molecule, may act as competitive inhibitor of the SAM-dependent methyl transferase reaction of the psiM enzyme, thus we postulated that it is necessary to increase cell capabilities of hydrolysis of S- adenosyl-l-homocysteine. To test this hypothesis, yeast strains having psiH, psiK, psiM, CPR genes from Psilocybe genus and an additional copy of the endogenous P. pastoris SAH1 (Adenosylhomocysteinase) gene integrated into the genome were generated. All 5 genes are placed under a GAP promoter. SAH1 is responsible for hydrolysis of S-adenosyl-l- homocysteine to L-homocysteine, that is recycled to SAM, and adenosine. Specifically, the P. pastoris strain CBS7435 was transformed with the vector pPpGAP-KanS- HIS4-psiM (P. cyan.)-psiK (P. cub.)-psiH (P. cyan.)-CPR (P. cyan.)-SAH1 (P. p.). A vector map is shown in Figure 2. Eight transformants were selected and analyzed by SDS-PAGE (results shown in Figure 3). The CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_8 strain was chosen for further work. We tested the CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_8 strain produced and found that it allowed full conversion of 5 µM of tryptamine per 1 g of WCW in 3 hours constantly without decrease of methylation rate as it was observed in similar strain without additional yeast SAH1 gene. Also, to increase total amount of synthesized psilocybin in yeast, amount of yeast WCW at the beginning was increased to 400 g/L from 200 g/L, because it takes only 8 hours of feeding to double the yeast biomass but allows to double the amount of psilocybin synthesis. In this setup, using CBS7435_pPpGAP- HIS4-psiM-psiK-psiH-CPR- SAH1_8 strain, conversion rate is 2 mM of tryptamine to 2 mM psilocybin per 3 hours - 570 mg of psilocybin is synthesized in 3 hours per 1L of medium, or approx.4.5 gr of psilocybin is synthesized in 24 hours after 8 batches of 2mM tryptamine added. Most of it remains in yeast cells and about 15-20 % is found in the yeast growth media. Also, the conversion can be extended further – we’ve achieved 6-7 g/L psilocybin titer, but in this case more psilocybin (25- 30 %) is found in the yeast growth media. Additionally, we prepared and tested a yeast strain with psiH, psiK, psiM, CPR genes from Psilocybe genus and both SAM2 from S. cerevisae and additional copy of P. pastoris SAH1 integrated in the genome. All 6 genes were placed under a GAP1 promoter. Cultivation of CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAM2-SAH1_7 strain didn’t show any improvements compared to CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_8 strain. Even to the contrary, addition of equal amount of methionine as tryptamine during cultivation of CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAM2-SAH1_7 strain showed decrease in methylation rate that was evident by the accumulation of Baeocystin and Norbaeosystin. We postulate that this is due increased synthesis of SAM and thus accumulation of S-adenosyl-L- homocysteine which inhibits function psiM enzyme. HPLC analysis The HPLC analysis referred to herein is standard HPLC gradient separation, performed with the following conditions: Column C182.6 µm 50 x 3 mm Solvent A 0.1% trifluoroacetic acid (TFA) in water Solvent B 0.1% TFA in methanol Flow rate 0.6 ml/min. Injection volume 8µl Gradient formation 5% B to 50 % B in 30 min. Column temperature 30 °C. Figure 4 shows the HPLC analysis of extract of yeast cells (CBS7435_pPpGAP- HIS4-psiM- psiK-psiH-CPR-SAH1_8) converting tryptamine to psilocybin. Elution times of metabolites (Fig. 4): Norbaeocystin – 3.9 - 4 min. Baeocystin – 5.4 – 5.5 min. Psilocybin – 6.6 – 6.7 min. Psilocin – 13.9 – 14 min. Tryptamine 16.9 – 17 min. Peak no. Time Area Height Width Area% Symmetry 1 1.983 285.2 30.8 0.1402 4.227 0.955 2 2.15 360.6 33.3 0.1443 5.344 0.502 3 2.366 144.2 27.7 0.0827 2.137 1.068 4 2.581 99.4 14.9 0.1136 1.473 3.18 5 2.661 88.7 15.2 0.0841 1.315 1.041 6 2.777 563.4 130.5 0.0644 8.351 0.567 7 3.049 77.2 7.5 0.1402 1.145 0.339 8 3.556 76.4 4.8 0.2047 1.132 0.712 9 4.017 34.3 4 0.1392 0.508 0.718 Table 1 – Results of HPLC analysis shown in Figure 4. Protocol for psilocybin biosynthesis Based on the above work, the inventors have developed the following protocol for psilocybin biosynthesis in yeast P. pastoris in 1L of BSM media. 1. Inoculate 100 ml shake flask with 20 ml of YEPD media with P. pastoris CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_8 strain (either from the research cell bank or from the Petri dish with the freshly grown cells). Incubate at 28˚C at 220 rpm for 22- 24 hours. OD600 after cultivation – 30-40. 2. Prepare 1L of BSM media (63 g/L glycerol, 0.45 g/L CaSO4, 7.3 g/L K2SO4, 5.8 g/L Mg2SO4x7H2O, 9 g/L NH4SO4). Aliquot 100 ml of BSM media in to the 500 ml volume shake flask for preparing inoculate. Use the rest of the media to prepare bioreactor (5L total volume Biostat A plus bioreactor was used). 3. To 100 ml of BSM media add 5 ml HMP (300 g/L), 0.4 ml PTM Trace Salts (Table 4) and YEPD media (20 g of bacto peptone and 10 g yeast extract in 950 mL water, with 50 mL of 40% glucose) with cultivated CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_8 strain to 0.2 OD600. Incubate at 28˚C at 220 rpm for 16-18 hours. OD600 after cultivation – 12-16. 4. Inoculate bioreactor with 100 ml of BSM media with cultivated CBS7435_pPpGAP -HIS4- psiM-psiK-psiH-CPR-SAH1_8 strain. Set the following parameters to be controlled in the bioreactor: 28˚C, pH 5.0, 1 vvm, 400 rpm, 30% DO. pH is controlled by addition of 28% NH4OH or 40% H3PO4. DO is controlled by stirrer (400-900 rpm) or addition of pure oxygen (0-100 %) cascades. 5. After 20-23 hours glycerol is consumed, evident by sudden increase in DO (DO spike). WCW at this point is 160-180 g/L. 6. Set pH to 7.0 and start glycerol feeding. Feed 50% glycerol (12 ml/L PTM1) starting from 4.8 ml/L/h rate that is constantly increased to 9.6 ml/L/h rate per 8 hours. 9.6 ml/L/h rate is constant for further process. 7. Prepare 320 ml 50mM tryptamine stock (in water if tryptamine HCL is used, or in 100 mM KH2PO4 if tryptamine is used). Filter sterilize. 8. After 8 hours of feeding set pH to 7.5. WCW at this point is 380-400 g/L. 9. Add 2 mM of tryptamine batch (40 ml of 50 mM tryptamine stock to 1L of media) every 3 hours for 8 additions. 16 mM of tryptamine is added in total per 24 hours resulting in 4.5 g/L of psilocybin if fully converted. 10. Extraction of Psilocybin after full conversion can be done in 2 ways: a) Add 30% of acetonitrile of total cell broth volume to bioreactor and mix for 60 min. Centrifuge the whole mixture for 5 min. 3000xg. Take supernatant, it contains approx. 90 % of synthesized Psilocybin. This method for psilocybin extraction is more advantageous because it is quicker, allows extraction of most of psilocybin but, because of the salts in the growth media, requires nano-filtration to transfer psilocybin in the suitable buffer (1 mM NaOH) in order to bind psilocybin to Sepharose Q resin. b) Centrifuge yeast cells for 5 min at 2000 x g. Remove growth media. Wash cell with dionized water, centrifuge for 5 min at 2000 x g. Extract psilocybin from yeast cell by suspending yeast cells in 1mM NaOH/30% acetonitrile solution (5 ml of solution to 1 g of biomass). Stir the suspension for 60 min and centrifuge for 5 min at 2000 x g. Take supernatant, it contains approx. 50-60 % of synthesized psilocybin (30-35 % of total psilocybin is in the growth media and in water, that was used for cell wash). Supernatant can be directly loaded on the Sepahrose Q column. This method for psilocybin purification can be used to avoid the need for nanofiltration. Extraction of psilocybin from yeast cells after fermentation A method for efficient extraction of psilocybin from yeast cells after fermentation was developed. Various buffers, solutions, yeast disruption methods and solution to WCW ratios were analyzed. Recovered amount of psilocybin from yeast cells and impact for further purification process were evaluated. Water-based buffers and solutions were found to have very low recovery even after disruption of yeast cells. Also, the disruption of yeast cells was found to have a negative impact for development of further purification processes due to large amount of cell-based impurities that are released after cell lysis, mostly proteins and nucleic acid. Thus, different organic solvents were screened for extraction of Psilocybin from undisrupted cells. Methanol and acetonitrile were found to have the best properties for extraction of psilocybin. Different concentrations (from 10 to 50 %) of acetonitrile and methanol in 1mM NaOH solution were tested. It was found that concentrations of 20%, 40%, and 50% all work. However, 30% of acetonitrile in 1mM NaOH recovers about 80-90% of psilocybin from undisrupted yeast cells. Also, it allows for simple further purification of psilocybin. Purification of psilocybin from extract of yeast cells Purification of psilocybin from the extract of the yeast cells was developed using strong anion exchange sorbent Sepharose Q HP. Anion exchange media was chosen because psilocybin has a negatively charged phosphate group. As most of the other negatively charged ions bind to positively charged functional groups of Sepharose Q HP media much more strongly than the phosphate ion, we chose to load Sepahrose Q HP with hydroxyl ions using 1M of NaOH, because hydroxyl group has lowest relative selectivity to anion exchangers. In order to be able to bind to Sepahrose Q HP, psilocybin must be in almost ion free solution and have a pH above 10.5 so that charge of dimethylamine nitrogen is neutral instead of positive. This was achieved by washing yeast cells with water and extracting psilocybin with 30% of acetonitrile in 1mM NaOH and adjusting pH of extract to 10.5 with NaOH. This setup allowed a binding capacity of 0.5 mg of psilocybin in yeast cell extract to 1 ml of Sepahrose Q HP media. For elution of bound psilocybin from anion exchanger several ions were tested, but the best selectivity was achieved using increasing hydroxyl ions gradient. This allowed elution of psilocybin into single peak (as shown in Figure 5 fractions 16-17) at 13-14 mS/cm conductivity with 90-95% psilocybin purity. For the second purification step, those fractions were combined, diluted 2x times with 1mM NaOH and again loaded on the Sepahrose Q HP column, washed with 1M NaOH and calibrated with 1mM NaOH. In this instance psilocybin bound to the column much more tightly – psilocybin was eluted at 29-39 mS/cm conductivity in much broader peak compared with the first Sepharose Q HP step (Figure 6 fractions 22-32). Psilocybin purity in all fractions (22-32) in this peak was 98-99%, with only single one impurity – baeocystin (Figure 7). Interestingly, as seen in Figure 6 the peak is double, but HPLC analysis of all fractions (22 to 32) showed no difference in psilocybin purity, so the reason of peak doubling is unknown. Peak no. Time Area Height Width Area% Symmetry 1 5.408 25.2 3.4 0.111 2.608 0.867 2 6.638 941.9 116.3 0.1225 97.392 0.821 Table 2 – Results of HPLC analysis shown in Figure 7. An example purification methodology can therefore be summarized as follows: 1. After conversion of substrate to psilocybin sediment cells by centrifugation at 1000 x g for 5 min. 2. Wash cells with MiliQ water (resuspend cells in water and sediment again). 3. Resuspend cells in 30% acetonitrile in 1mM NaOH at 1 to 5 ratio (5 ml of solution to 1 gram of wet cell weight). 4. Stir the mixture for 30-60 min. 5. Centrifuge the mixture for 5 min. at 3000 x g. Collect the solution (extract) and discard the cells. Adjust pH of extract to 10.5 with NaOH. 6. Prepare the column by washing with 5 column volumes of 1M NaOH and subsequently calibrating with 5 column volumes of 1mM NaOH. 7. Load the extract on to the Sepharose Q HP column of necessary volume at 50-60 cm/h flow velocity. 8. Wash the column with 3 column volumes of 1mM NaOH at the same flow velocity. 9. Run the gradient of 5 column volumes to 150 mM NaOH at 50-60 cm/h flow velocity. Collect 1/10 column volume fractions. 10. Analyze fractions by HPLC. Psilocybin elutes in a single peak at about 65-70 mM NaOH 13-14 mS/cm. 11. Combine fraction with satisfactory psilocybin purity and dilute 2x fold with 1 mM NaOH. 12. Prepare the Sepharose Q HP column by washing with 5 column volumes of 1M NaOH and subsequently calibrating with 5 column volumes of 1mM NaOH. 13. Load the combined and diluted fractions on to the Sepharose Q HP column at 50-60 cm/h flow velocity. 14. Wash the column with 3 column volumes of 1mM NaOH at the same flow velocity. 15. Run the gradient of 5 column volumes to 300 mM NaOH at 50-60 cm/h flow velocity. Collect 1/10 column volume fractions. 16. Analyze fractions by HPLC. Psilocybin elutes in a peak at about 220-260 mM NaOH 30-38 mS/cm. 17. Combine fraction with satisfactory psilocybin purity. Add 20 mM TRIS pH 7.5 and adjust pH to 7.5 with HCl. Lyophilize or store at -20˚C or -80˚C. Example 2 – Biosynthesis of ({3-[2-(dimethylamino)ethyl]-1-benzothiophen-4- yl}oxy)phosphonic acid (“S-psilocybin”) in the yeast P. pastoris cells using enzymes from fungi of Psilocybe genus In this Example ({3-[2-(dimethylamino)ethyl]-1-benzothiophen-4-yl}oxy)phosphonic acid (Substance (5) below – “S-psilocybin”) is synthesized from 2-(benzothien-3-yl)ethylamine (Substance (1) below – “S-tryptamine”) via the intermediates (Substances (2), (3) and (4) below) using the same psiH, psiK and psiM enzymes as in Example 1. (A dephosphorylated form of the product is shown as Substance (6) (“S-psilocin”) below.) Substance 1 Name (IUPAC) 2-(benzothien-3-yl)ethylamine hydrochloride
Figure imgf000033_0001
Substance 2
Figure imgf000033_0002
Figure imgf000033_0003
Figure imgf000033_0004
Substance 5 Formula C12H16NO4PS MW 301.2988
Figure imgf000034_0001
Figure imgf000034_0002
Fermentation technology a) Collection of yeast P. pastoris strains with integrated into the genome psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus having different S-tryptamine (Substance (1)) to S-psilocybin (Substance (5)) conversion properties were screened to find a strain with the best conversion rate. Target genes were placed under strong constitutive GAP promoter (enzymes are produced constantly) or very strong inducible AOX1 promoter (enzymes are produced only when methanol is added). Our research has shown that currently the best option is P. pastoris strain, where target genes are placed under GAP promoter. It gives several advantages: (i) amount of synthesized enzymes is optimum for conversion rate/speed of S-tryptamine to S-psilocybin without depleting cellular metabolites necessary for enzymatic reactions; (ii) there is no need to use methanol, which is toxic, requires safety protocols and equipment – makes technology greener; (iii) no oxidative stress in cells, which is caused by methanol metabolism in yeast, allows to avoid forming of dephosphorylated S-psilocybin (Substance (6) above – “S-psilocin”) polymers. Strain collection used for the research: I. psiH, psiK, psiM and CPR genes under GAP promoter in P. pastoris CBS7435 strain: CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1; CBS7435_pPpGAP- HIS4-psiM-psiK-psiH-CPR_2, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH- CPR_5, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_8. II. psiH, psiK, psiM and CPR genes under AOX1 promoter CBS7435_pPpAOX-HIS4- psiM-psiK-psiH-CPR_1_MutS (MutS phenotype – slow methanol utilization), CBS7435_pPpAOX-HIS4-psiM-psiK-psiH-CPR_2_Mut+ (Mut+ phenotype – fast methanol utilization). Materials and methods used for strain screening: I. P. pastoris CBS7435 strain. pPpKanS-HIS4, pPpGAP-HIS4 vectors, prepared in this research. Chemical synthesis of psiH (from P. cyanenscens), psiK (P. cubensis), psiM (P. cyanenscens) and CPR (P. cyanenscens) was ordered from GenScript. II. Classical genetic and microbiological manipulations for construction of necessary expression vectors, transformation of P. pastoris and selection of yeast strains with optimal number of integrated target genes. III. Selected yeast strains were cultivated in the Basal salt media (BSM) with glycerol (see Tables 3 and 4 below) for 24 hours. After 24 hours the either additional 2% of glycerol were added to CBS7435_pPpGAP strains or either 1% (Mut+) or 0.5% (MutS) of methanol for CBS7435_pPpAOX strains and supplemented with 1 mM of S-tryptamine. IV. Metabolites were extracted by adding equal volume of acetonitrile to whole cell broth (WCB). Mixture was vortexed for 2 min. and centrifuged for 5 min. at 3000 x g. V. Extracts were analyzed by HPLC (discussed in next section). Glycerol 98% 20 g Milli Q ultrapure water (UPW) 912 g Calcium sulfate dihydrate 0.46 g Magnesium sulfate heptahydrate 5.84 g Potassium sulfate 7.34 g Ammonium sulfate 9.0 g Hexametaphosphate 300g/L solution 84 ml Trace metal solution (PTM) 4 ml Table 3 – BSM media, 1 Litre Copper (II) sulfate 3.0 g Sodium iodide 0.040 g Manganese (II) sulfate 1.50 g Sodium molybdate 0.100 g Boric acid 0.010 g Cobalt (II) chloride 0.250 g Zinc chloride 10.0 g Iron (II) sulfate heptahydrate 32.5 g Biotin 0.100 g Sulfuric acid, conc. 2.50 ml Table 4 - PTM, 1 Litre The HPLC analysis referred to in V. is standard HPLC gradient separation, performed with the following conditions: Column C182.6 µm 50 x 3 mm Solvent A 0.1% trifluoroacetic acid (TFA) in water Solvent B 0.1% TFA in methanol Flow rate 0.6 ml/min. Injection volume 8µl Gradient formation 5% B to 50 % B in 15 mins. Column temperature 30 °C. HPLC results alongside mass spectra for Substance (4) and for Substance (5) are shown in Figures 8A and 8B, and Figures 9A and 9B, respectively. b) P. pastoris fermentation conditions, that allow uptake of S-tryptamine to the cell, and efficient conversion to S-psilocybin were developed, that allow complete conversion of 2mM of S-tryptamine to S-psilocybin resulting in 600 mg of S-psilocybin in 1L WCB. The process uses BSM media as shown below in Table 5. Glycerol 98% 63 g Milli Q ultrapure water (UPW) 912 g Calcium sulfate dihydrate 0.46 g Magnesium sulfate heptahydrate 5.84 g Potassium sulfate 7.34 g Ammonium sulfate 9.0 g Hexametaphosphate 300g/L solution 84 ml Trace metal solution (PTM) 4 ml Table 5 – BSM media, 1L Bioreactors: EDF-1.2 bioreactors (Biotechniskais centrs, AS) Starting fermentation conditions: 28˚C, 1 vvm air, pH 5.0, 400 rpm. 30% DO (dissolved oxygen) is maintained by increasing stirrer speed (400-1200 rpm) and addition of oxygen (0-100%) into the air flow. pH is maintained by automatic addition of 40% phosphoric acid and 28% ammonium solution. After glycerol depletion (manifested by DO spike), pH is increased to 7.3 and 50% glycerol solution feeding at 10ml/h/L is initiated. After 1h of feeding 2mM of S-tryptamine is added (50mM stock of 2-(benzothien-3-yl)ethylamine hydrochloride is prepared in water and filter- sterilized). After 7-8 hours full conversion of S-tryptamine to S-psilocybin is completed, resulting in approx. 600 mg S-psilocybin in 1L of WCB. About 20-25% of S-psilocybin is in the culture media and the rest is inside the yeast cells. Under normal yeast fermentation conditions, pH 5.0, S-tryptamine is relatively uncharged molecular that can’t freely cross yeast cells plasma membrane. Thus, we developed a fermentation media, that allows yeast cell growth under pH 7 – 7.8. Under such pH S- tryptamine is partly charged and can cross plasma membrane. Most of the phosphate salts used for yeast fermentation precipitate under pH 7 – 7.8. Instead of common phosphate salts or phosphoric acid we use sodium hexametaphosphate (HMP) as phosphate source. It allows to grow P. pastoris in wide range of pH. S-tryptamine conversion to S-psilocybin is 10 µM of S- tryptamine to S-psilocybin in 6-7 hours per 1 gram of wet cell weight (WCW) by CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1 strain. Average amount of WCW during conversion is about 200-240 g/L, so conversion rate is 2 mM of S-tryptamine to 2 mM S- psilocybin per 6-7 hours (600 mg of S-psilocybin is synthesized in 6-7 hours). After full conversion of 2mM of S-tryptamine additional 2mM of substrate can be added to further increase S-psilocybin yield. Using such setup, about 2.4 gr of S-psilocybin per 1 L of yeast fermentation media can be produced in 24 hours. Most of it remains in yeast cells and about 20-25 % is found in yeast growth media. Materials and methods used for the development of fermentation technology: I. P. pastoris CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1 strain. II. Different pH values, glycerol feeding profiles, amount and addition time of substrate Substance (1) - (S-tryptamine) were analyzed to develop the optimal fermentation conditions for efficient and complete conversion of substrate to final product - Substance (5) (S-psilocybin). Purification technology Testing was performed to achieve efficient and cost-effective extraction and purification of S- psilocybin. Materials and methods used for S-psilocybin extraction optimization: I. Yeast cell biomass after fermentation containing 1,5-2 mg of S-psilocybin per gram of WCW. II. S-psilocybin was extracted by resuspending cells in 1:1 to 1:5 g of WCW to ml of solution ratio using different solutions. Mixture was vortexed for 2 min. and centrifuged for 5 min. at 3000 x g. III. For yeast cell disruption equal volume of glass beads were added. IV. Extracts were analyzed by HPLC. Materials and methods used for S-psilocybin chromatographic purification optimization: I. Extract of yeast cells, containing S-psilocybin, using 30% of acetonitrile in 1mM NaOH. II. Classical chromatography techniques were used to determine optimal S-psilocybin binding conditions, binding capacity and elution profiles. III. Fractions were analyzed by HPLC. It was noted that the findings described above in Example 1 in relation to the extraction and purification of psilocybin also applied to S-psilocybin. Accordingly, the purification methodology points 1 to 17 set out in Example 1 can also be used. S-psilocybin elutes as a single peak at about 70-75 mM NaOH 12.7-12.9 mS/cm (Figure 10, fractions 33-34). This Example demonstrates the ability of the method of the invention to produce target alkaloids that are related to psilocybin. Example 3 – Biosynthesis of 3-(N,N-dimethylaminoethyl)benzo[b]furan-4-yl phosphate (O-Psilocybin) in the yeast P. pastoris cells using enzymes from fungi of Psilocybe genus In this Example 3-(N,N-dimethylaminoethyl)benzo[b]furan-4-yl phosphate – “O-psilocybin” (Substance (5) below) is synthesized from 2-(benzofuran-3-yl)ethylamine – “O-tryptamine” (Substance (1) below) via the intermediates (Substances 2 to 4 below) using the same psiH, psiK and psiM enzymes as in Example 1. (A dephosphorylated form of the product is shown as Substance 6 (“O-psilocin”).) Substance 1 Name (IUPAC): 2-(benzofuran-3-yl)ethylamine hydrochloride Formula: C10H11NO*ClH MW: 197.664 CAS no. 27404-32-6
Figure imgf000039_0001
Substance 2 Name: 3-(2-aminoethyl)-1-benzofuran-4-ol Formula: C10H11NO2 MW: 177.20 CAS no. 1890736-76-1
Figure imgf000039_0002
Figure imgf000039_0003
Substance 4
Figure imgf000039_0004
Substance 5
Figure imgf000040_0001
Substance 6
Figure imgf000040_0002
Fermentation technology a) Yeast P. pastoris strain (CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR-SAH1_8), that previously was used for psilocybin, with integrated into the genome psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus and yeast SAH1 gene (all genes placed under strong constitutive GAP promoter – such that enzymes are produced constantly), was used for biosynthesis of O-Psilocybin. b) The same P. pastoris fermentation conditions, that allow efficient biosynthesis of psilocybin and S-psilocybin were used, that allow complete conversion of 2mM of O-tryptamine to O- psilocybin resulting in approx. 570 mg of O-psilocybin in 1L WCB in 7-8 hours (as shown in Figures 11A to 11C). Most of it remains in yeast cells and about 30-35 % is found in yeast growth media. (Figure 11A elution times of metabolites: O-norbaeocystin – undetected; O- baeocystin – 10.0-10.1 min; O-psilocybin – 10.6-10.7 min; O-psilocybin – 15.7-15.8 min; 2- Benzofuran-3-yl)ethylamine 15.6-15.7 min.) Purification technology Purification of O-psilocybin from yeast cells was done according to previously developed method for psilocybin purification. 1. After conversion of substrate to O-Psilocybin sediment cells by centrifugation at 1000 x g for 5 min. 2. Wash cells with MiliQ water (resuspend cells in water and sediment again). 3. Resuspend cells in 30% acetonitrile in 1mM NaOH at 1 to 5 ratio (5 ml of solution to 1 gr of wet cell weight). 4. Stir the mixture for 60 min. 5. Centrifuge the mixture for 5 min. at 3000 x g. Collect the solution (extract) and discard the cells. Adjust pH of extract to 10.5 with NaOH. 6. Prepare the column by washing with 5 column volumes of 1M NaOH and subsequently calibrating with 5 column volumes of 1mM NaOH. 7. Load the extract on to the Sepharose Q HP column of necessary volume at 50-60 cm/h flow velocity. 8. Wash the column with 3 column volumes of 1mM NaOH at the same flow velocity. 9. Run the gradient of 5 column volumes to 150 mM NaOH at 50-60 cm/h flow velocity. Collect 1/10 column volume fractions. 10. Analyze fractions by HPLC. O-Psilocybin elutes in a single peak at about 40-50 mM NaOH 7-8 mS/cm. Fractions 6-9 in Figure 12. 11. Combine fractions with satisfactory O-Psilocybin purity and dilute 2x fold with 1 mM NaOH. 12. Prepare the Sepharose Q HP column by washing with 5 column volumes of 1M NaOH and subsequently calibrating with 5 column volumes of 1mM NaOH. 13. Load the combined and diluted fractions on to the Sepharose Q HP column at 50-60 cm/h flow velocity. 14. Wash the column with 3 column volumes of 1mM NaOH at the same flow velocity. 15. Run the gradient of 5 column volumes to 300 mM NaOH at 50-60 cm/h flow velocity. Collect 1/10 column volume fractions. 16. Analyze fractions by HPLC. O-Psilocybin elutes in a peak at about 130-150 mM NaOH 23-26 mS/cm. . Fractions 11-13 in Figure 13. 17. Combine fraction with satisfactory O-Psilocybin purity. Add 20 mM sodium phosphate and adjust pH to 7.5 with HCl. Lyophilize or store at -20˚C or -80˚C. Such purification method allowed purification of O-psilocybin from yeast cells to 92% purity (see Figure 14). Peak no. Time Area Height Width Area% Symmetry 1 10.543 6225.7 1156.8 0.0828 92.012 0.969 2 13.42 115.2 18.7 0.0898 1.702 0.682 3 15.501 425.3 83.9 0.0771 6.286 0.886 Table 6 – HPLC results shown in Figure 14. This Example further demonstrates the ability of the method of the invention to produce target alkaloids that are related to psilocybin. Example 4 – Steps for biosynthesis of further compounds in the yeast P. pastoris cells using enzymes from fungi of Psilocybe genus Example 4A – To provide a compound according to formula I described herein where X is N and Z is C and W is N (a benzimidazole derivative). The substrate (starting material) can be 2- (1H-Benzimidazol-1-yl)ethylamine hydrochloride and the following synthesis pathway can be used: 1. Substrate (starting material) 2-(1H-Benzimidazol-1- yl)ethylamine hydrochloride 2. 3. 4. 5. 6. Example 4B – To provide a compound according to formula I described herein where X is O and Z is N and W is C (a benzisoxazole derivative). The substrate (starting material) can be 1,2-Benzisoxazole-3-ethanamine and the following synthesis pathway can be used: Example 4C – To provide a compound according to formula I described herein where X is O and Z is C and W is N (a benzo[d]oxazole derivative). The substrate (starting material) can be 2-(benzo[d]oxazol-3(2H)-yl)ethan-1-amine and the following synthesis pathway can be used:
Figure imgf000045_0001
Figure imgf000045_0002
Figure imgf000045_0003
Figure imgf000045_0004
Figure imgf000046_0001
FUNCTIONAL STUDIES The functional activity of the psilocybin related compounds described herein can be shown in vitro using radioligand binding assays and in vivo using head twitch response studies. Competition Assay Competition assays can be used to show binding to 5-HT receptors, e.g. 5-HT1A. A suitable example assay is described by Gifford Bioscience Limited (www.giffordbioscience.com - Radioligand Binding Assay Protocols). This assay is as follows: Membrane preparation: Frozen tissue or washed cells are homogenized in 20 volumes of cold lysis buffer (50mM Tris-HCl, 5 mM MgCl2, 5 mM EDTA, protease inhibitor cocktail). After a low speed spin (100 x g for 3 minutes) to remove large tissue chunks (tissue homogenates), the homogenate is centrifuged at 17,000 x g for 10 minutes at 4 °C to pellet the membranes. The pellet is resuspended in fresh buffer and centrifuged at the same speed for a second time, again at 4 °C. The pellet is then resuspended into buffer (15 ml) containing 10% sucrose as a cryoprotectant, divided into 1 ml aliquots and stored at -80 °C. A sample of the homogenate is analyzed for protein content. On the day of the assay the membrane preparation is thawed and the pellet resuspended in final assay binding buffer (50 mM Tris, 5 mM MgCl2, 0.1 mM EDTA, pH 7.4). Incubation and filtration: The filtration binding assay is carried out in 96-well plates in a final volume of 250 µL per well. To each well is added 150 µL membranes (3 - 20 µg protein for cells or 50 - 120 µg protein for tissue), 50 µL of the competing test compound and 50 µL of radioligand solution in buffer. The plate is incubated at 30 °C for 60 minutes with gentle agitation. The incubation is stopped by vacuum filtration onto 0.3% PEI presoaked GF/C filters using a 96-well FilterMate™ harvester followed by four washes with ice-cold wash buffer. Filters are then dried for 30 minutes at 50 °C. The filter is sealed in polyethylene, scintillation cocktail (Betaplate Scint; PerkinElmer) added and the radioactivity counted in a Wallac® TriLux 1450 MicroBeta counter. Data analysis: For each drug concentration, non-specific binding is subtracted from total binding to give specific binding. Data is fitted using the non-linear curve fitting routines in Prism® (Graphpad Software Inc). For competition assays, Ki values are calculated from IC50 values using the formula Ki = IC50 / (1 + ([S]/Kd)) where [S] is the radiotracer concentration used in the assay and Kd is the dissociation constant of the radiotracer. Saturation Assay Saturation assays can also be used to show binding to 5-HT receptors, e.g. 5-HT1A. A suitable example assay is described by Gifford Bioscience Limited (www.giffordbioscience.com - Radioligand Binding Assay Protocols). This assay is as follows: Membrane preparation: as described above for the competition assay. Incubation and filtration: The filtration binding assay is carried out in 96-well plates in a final volume of 250 µL per well. To each well is added 150 µL membranes (3 - 20 µg protein for cells; 50 - 120 µg protein for tissue), 50 µL of the unlabeled compound (non-specifics) or buffer and 50 µL of radioligand solution in binding buffer. The radioligand is added at up to 8 different concentrations (e.g. 0.2 - 20 nM). The plate is incubated at 30 °C for 60 minutes with gentle agitation. The incubation is stopped by vacuum filtration onto 0.3% PEI presoaked GF/C filters using a 96-well FilterMate™ harvester followed by four washes with ice-cold wash buffer. Filters are then dried for 30 minutes at 50 °C. The filter is sealed in polyethylene, scintillation cocktail (Betaplate Scint; PerkinElmer) added and the radioactivity counted in a Wallac® TriLux 1450 MicroBeta counter. Data analysis: For each radioligand concentration, non-specific binding is subtracted from total binding to give specific binding. Bound CPM values are converted to fmoles per mg protein. Data is fitted using the saturation analysis non-linear curve fitting routines in Prism® (Graphpad Software Inc). The Kd (in nM) and Bmax (fmol/mg or sites/cell) are derived from the saturation curve. Head Twitch Response Studies Head twitch responses are considered a valuable indicator of serotonergic activity and have been extensively used to evaluate compounds with potential psychoactive or neuromodulatory effects. Animal Model: C57BL/6J male mice aged 8-12 weeks are used in the study. Water and food are provided ad libitum throughout the entire study. The holding room maintains a temperature of 21±1°C, humidity at 55±10%, and operates on a 12-hour light/dark cycle. All efforts are made to minimize animal suffering and reduce the number of animals used. Each animal are weighed at the start of the experiment. Treatment Groups: Mice are randomly assigned to different treatment groups, with each group receiving either a PSY series compound or a control substance (saline). Preparation of Test Substances: Stock solutions of test substances are prepared on the same day of the experiment and are protected from direct light. Administration: The compounds are administered via intraperitoneal injections (i.p.), and the dosages administered are individually determined based on each animal's body weight. Behavioural Assessment: Immediately following the intraperitoneal injection of the drug, a mouse is placed in the open field, and video recording is initiated for 30 minutes. A camera is positioned directly above the mouse to capture its behaviour. The environment is kept quiet to minimize disturbances during the recording period. Data Collection: Review the recorded videos and count the number of head twitch responses displayed by each mouse during the observation period. A head twitch response is characterized by a rapid, involuntary, rotational movement of the head. Data Analysis: Statistical analyses are conducted to evaluate the significance of head twitch responses induced by psilocybin related compounds in comparison to the control group. The one-way analysis of variance (ANOVA) method is employed for this analysis. Example of 5-HT2A Serotonin Receptor Agonist Assay Abbreviations used in example: SD – standard deviation; DMEM – Dulbecco’s Modified Eagle’s Medium; PBS - Phosphate buffered saline; FBS – Fetal Bovine Serum; and a.u. – arbitrary units. The aim of the present example was to screen the agonist effect of 4 test compounds on the 5- HT2A serotonin receptor activity using the U2OS 5-HTR2A Serotonin receptor Hitseeker cell line. HiTSeeker 5-HTR2A/U2OS contains U2OS cells stably expressing human 5-HT2A Serotonin receptor with no tag. The HiTSeeker HTR2A cell line has been designed to assay compounds or analyse their capability to modulate 5-HT2A Serotonin receptor. When the agonist binds to HTR2A a G protein is activated, which in turn, triggers a cellular response mediated by second messengers (Calcium). This cellular response can be measured quantifying calcium increase inside the cell determining the intensity of Fluo4. Materials and Method Reagents and equipment: - U2OS cells (CLS gmbh 300192) - DMEM (Sigma-Aldrich D6429) - FBS (Sigma-Aldrich F7524) - Flat bottom black 96-well plates (Perkin Elmer 6005182) - Fluo-4 NW Calcium Assay Kits (ThermoFisher F36206) - Serotonin, 5-HT (Sigma H9523) Assay Development: The modulatory effect of the compounds was analyzed measuring the Calcium increase using Fluo-4 NW assay. When calcium concentration increases calcium binds to Fluo-4 increasing the fluorescence at 494 nm. Fluorescence intensity acquisition was performed in the Synergy™ 2 Multi-Detection microplate reader from Biotek. Vehicle (NaCl or H2O) was used as negative control; 5-HT was used as positive control. Compounds were tested at 200, 100, 50, 25, 10, 5, 2.5, 1, 0.5 µM in triplicates. Method: Day 1. The U2OS 5-HTR2A Hitseeker cell line was thawed (2x106 cells per T25). Day 2. The cells were maintained in DMEM-F12 supplemented with 10% FBS at 37ºC in a humidified 5% CO 2 atmosphere. Day 3. The cells were plated at a concentration of 20.000 cells/well (+/-1000 cells) in 96- well plates. Cells were maintained in DMEM-12 medium supplemented with 10% FBS during 48h at 37ºC in a humidified 5 % CO 2 atmosphere. Day 5. To detect calcium increase, the cells were stained with Fluo-4 NW during 1 hour (followed manufacturer protocol). Subsequently, the compounds were added, and fluorescence was measured immediately. To detect Fluo-4 the filters used were 480/20 and 528/20 nm for excitation and emission, respectively. Excel 2016 and Sigmaplot 11 were used for data management. Structures of Compounds: PSY-1342 PSY-1343 PSY-1185 PSY-1186 Test compounds were dissolved and tested at nine concentrations: 200, 100, 50, 25, 10, 5, 2.5, 1 and 0.5 µM. 5-HT at 10 µM was used as positive control and vehicle (water for PSY- 1185 and PSY-1186 and NaCl for PSY-1342 and PSY-1134) as negative control. Figure 15 shows the results obtained after treating the cells with the compounds. Data are presented as fluorescence intensity of Fluo-4 representing calcium increase after HTR2A stimulation. For each compound also negative control (vehicle) and 5-HT are presented. Two compounds showed an increase in intensity compared to the control: compound PSY- 1185 is the most active, exhibiting an increase of 2.76 fold compared to the control and demonstrating a clear dose-dependent activity; and compound PSY-1343 exhibit a smaller increase, 2.4 fold, and only at higher concentrations, but in a concentration-dependent manner. Examining the results in Figure 1, dose-response curve of activity was generated for the PSY- 1185, not for PSY-1343 because activity do not reach the plateau (Figure 2). The values were normalized with respect to the negative control (0%), and 100% was set as the maximum activity of the compound. Curve was fitted to a sigmoidal curve, and the EC 50 value could be calculated using a 4-parameter sigmoidal model giving an EC 50 of 2.88x10-6 M. It is worth mentioning that there is considerable variability at the higher concentrations. Conclusions: Among the four compounds studied, PSY-1185 and PSY-1343 demonstrated agonist activity at the 5- HT2A receptor, though with varying degrees of potency. PSY-1185 emerged as the most potent activator, showing a clear dose-response relationship across the concentrations tested, with an EC 50 value of 2.88 x 10⁻⁶ M. This indicates that PSY-1185 has a relatively high affinity for the 5-HT2A receptor, making it the strong agonist. PSY- 1343 exhibited measurable activity, and this was only observed at the two highest concentrations tested. These findings demonstrate the capabilities of PSY-1185 and PSY-1343. SEQUENCES: The sequences allocated SEQ ID Nos. herein have the following sequences: SEQ ID No: 1 Description: cDNA sequence of cytochrome P450 reductase CPR (P. cyanescens) ATGGCTTCCAGTAGTTCCGATGTTTTTGTCTTAGGTTTGGGTGTTGTTTTGGCTGCT TTGTATATTTTTAGAGATCAATTATTTGCCGCATCAAAACCAAAAGTTGCTCCAGT TTCTACTACAAAACCAGCAAATGGTTCAGCTAACCCAAGAGATTTCATCGCTAAG ATGAAGCAAGGTAAAAAGAGAATCGTTATTTTCTATGGTTCTCAAACTGGTACAG CAGAAGAATACGCTATCAGATTGGCAAAGGAAGCTAAGCAAAAGTTCGGTTTGG CATCTTTAGTTTGTGATCCAGAAGAATACGATTTCGAAAAGTTGGATCAATTGCC AGAAGATTCAATTGCTTTCTTTGTTGTTGCTACTTACGGTGAAGGTGAACCAACA GATAACGCTGTTCAATTGTTGCAAAATTTGCAAGATGATTCATTCGAATTTTCTAA CGGTGAAAGAAAGTTGTCTGGTTTAAAGTACGTTGTTTTTGGTTTAGGTAATAAG ACTTACGAACATTACAATTTGATTGGTAGAACAGTTGATGCACAATTAGCTAAAA TGGGTGCTGTTAGAGTTGGTGAAAGAGGTGAAGGTGACGATGATAAGTCAATGG AAGAAGATTACTTGGAATGGAAAGATGGCATGTGGGATGCTTTTGCTGCAGCTAT GGGTGTTGAAGAAGGTCAAGGTGGTGACTCTGCAGATTTCGTTGTTTCAGAATTA GAATCTCATCCACCAGAAAAAGTTTATTTGGGTGAATACTCAGCAAGAGCTTTAA CTAAGACAAAGGGTATTCATGATGCTAAAAATCCATTGGCAGCTCCAATCACTGT TGCAAGAGAATTATTCCAATCTGTTGTTGATAGAAACTGTGTTCATGTTGAATTCA ATATCGAAGGTTCAGGTATCACATACCAACATGGTGACCATGTTGGTTTGTGGCC ATTGAACCCAGATGTTGAAGTTGAAAGATTGTTGTGTGTTTTGGGTTTAACTGAA AAGAGAGATGCTGTTATTTCAATCGAATCTTTAGATCCAGCATTAGCTAAAGTTC CATTTCCAGTTCCAACTACATATGCAGCTGTTTTGAGACATTACATTGATGTTTCT GCAGTTGCTGGTAGACAAATCTTGGGTACTTTGTCAAAGTTTGCTCCAACACCAG AAGCAGAAGCATTTTTGAAAAATTTGAACACTAATAAGGAAGAATACCATAACG TTGTTGCAAACGGTTGTTTGAAGTTGGGTGAAATCTTGCAAGTTGCTACTGGTAA CGATATTACTGTTGCACCAACACCAGGTAATACTACAAAGTGGCCAATCCCATTC GATATCATCGTTTCTGCTATCCCAAGATTGCAACCAAGATACTACTCAATCTCTTC ATCTCCAAAGGTTCATCCAAATACTATTCATGCAACAGTTGTTGTTTTGAAGTACG AAAACGTTCCAACTGATCCAATTCCAAGAAAATGGGTTTACGGTGTTGGTTCTAA TTTCTTGTTGAATTTGAAGCATGCTATTAATAAGGAACCAGTTCCTTTTATTACTC AAAACGGTGAACAAAGAGTTGGTGTTCCAGAATATTTGATTGCTGGTCCAAGAGG TTCATACAAGACAGAATCTCATTTCAAGGCACCAATCCATGTTAGAAGATCTACT TTTAGATTGCCAACAAACCCAAAGTCACCAGTTATTATGATTGGTCCAGGTACAG GTGTTGCTCCTTTTAGAGGTTTTGTTCAAGAAAGAGTTGCATTAGCTAGAAGATCT GTTGAAAAGAATGGTCCAGAATCATTGAACGATTGGGGTAGAATCTCTTTGTTTT ATGGTTGTAGAAGATCAGATGAAGATTTCTTGTACAAGGATGAATGGCCACAATA CCAAGAAGAATTGAAGGGTAAATTCAAATTGCATTGTGCTTTTTCTAGAGAAAAC TACAAGCCAGATGGTTCAAAGATCTATGTTCAAGATTTGATCTGGGAAGATAGAG AACATATCGCAGATGCTATCTTGAACGGTAAAGGTTACGTTTACATTTGTGGTGA GGCTAAGTCAATGTCTAAGCAAGTTGAAGAAGTTTTGGCAAGAATTTTAGGTGAA GCTAAAGGTGGTTCTGGTGCAGTTGAAGGTGTTGCCGAAATTAAGTTGTTGAAAG AAAGAAGTAGATTGATGTTAGATGTATGGAGTTAG SEQ ID No: 2 Description: amino acid sequence of cytochrome P450 reductase CPR (P. cyanescens) MASSSSDVFVLGLGVVLAALYIFRDQLFAASKPKVAPVSTTKPANGSANPRDFIAKM KQGKKRIVIFYGSQTGTAEEYAIRLAKEAKQKFGLASLVCDPEEYDFEKLDQLPEDSI AFFVVATYGEGEPTDNAVQLLQNLQDDSFEFSNGERKLSGLKYVVFGLGNKTYEHY NLIGRTVDAQLAKMGAVRVGERGEGDDDKSMEEDYLEWKDGMWDAFAAAMGVE EGQGGDSADFVVSELESHPPEKVYLGEYSARALTKTKGIHDAKNPLAAPITVARELFQ SVVDRNCVHVEFNIEGSGITYQHGDHVGLWPLNPDVEVERLLCVLGLTEKRDAVISIE SLDPALAKVPFPVPTTYAAVLRHYIDVSAVAGRQILGTLSKFAPTPEAEAFLKNLNTN KEEYHNVVANGCLKLGEILQVATGNDITVAPTPGNTTKWPIPFDIIVSAIPRLQPRYYS ISSSPKVHPNTIHATVVVLKYENVPTDPIPRKWVYGVGSNFLLNLKHAINKEPVPFITQ NGEQRVGVPEYLIAGPRGSYKTESHFKAPIHVRRSTFRLPTNPKSPVIMIGPGTGVAPF RGFVQERVALARRSVEKNGPESLNDWGRISLFYGCRRSDEDFLYKDEWPQYQEELK GKFKLHCAFSRENYKPDGSKIYVQDLIWEDREHIADAILNGKGYVYICGEAKSMSKQ VEEVLARILGEAKGGSGAVEGVAEIKLLKERSRLMLDVWS SEQ ID NO: 3 Description: cDNA sequence psiK (P. cubensis) ATGGCATTCGATCTAAAAACAGAAGATGGTCTAATTACATACTTGACAAAACATC TATCCTTGGATGTTGATACATCTGGTGTTAAAAGATTATCTGGTGGATTCGTTAAT GTTACATGGAGAATTAAACTGAACGCTCCATATCAAGGTCATACTTCTATTATTCT AAAGCATGCTCAACCACATATGTCTACTGATGAAGACTTCAAAATTGGTGTTGAA AGATCTGTCTATGAATATCAAGCTATTAAGTTGATGATGGCTAATAGAGAAGTTC TAGGTGGTGTTGATGGTATTGTTAGTGTTCCAGAAGGTCTAAATTATGATCTAGA AAATAACGCTCTGATCATGCAAGATGTTGGTAAAATGAAAACATTGTTGGATTAC GTTACCGCTAAACCACCATTAGCTACAGATATTGCTAGATTGGTTGGTACAGAAA TTGGTGGATTCGTCGCTAGATTACATAATATTGGTAGAGAAAGAAGGGATGATCC AGAATTCAAATTCTTCTCTGGTAATATTGTCGGTAGAACAACTTCTGATCAATTGT ATCAAACTATCATCCCAAATGCTGCTAAATATGGTGTTGATGATCCATTGTTGCCA ACAGTTGTTAAAGATCTAGTTGATGATGTTATGCATTCTGAAGAAACTCTAGTTAT GGCTGATCTATGGTCTGGTAATATATTGTTGCAATTAGAAGAGGGTAACCCATCT AAATTGCAAAAAATATATATCCTGGACTGGGAATTATGTAAATATGGTCCAGCTT CTCTAGATCTAGGTTACTTCTTAGGTGATTGTTATCTAATTAGCAGATTCCAAGAT GAACAAGTTGGTACTACTATGAGACAAGCATATCTACAATCTTATGCTAGAACTT CTAAGCATTCTATTAACTATGCTAAGGTTACAGCTGGTATTGCTGCTCATATTGTT ATGTGGACTGACTTCATGCAATGGGGTTCTGAAGAAGAAAGAATTAACTTCGTTA AGAAGGGTGTTGCTGCATTCCATGATGCTAGAGGTAATAATGATAATGGTGAAAT TACCTCTACCTTATTGAAAGAATCTTCTACAGCTTAA SEQ ID NO: 4 Description: amino acid sequence psiK (P. cubensis) MAFDLKTEDGLITYLTKHLSLDVDTSGVKRLSGGFVNVTWRIKLNAPYQGHTSIILKH AQPHMSTDEDFKIGVERSVYEYQAIKLMMANREVLGGVDGIVSVPEGLNYDLENNA LIMQDVGKMKTLLDYVTAKPPLATDIARLVGTEIGGFVARLHNIGRERRDDPEFKFFS GNIVGRTTSDQLYQTIIPNAAKYGVDDPLLPTVVKDLVDDVMHSEETLVMADLWSG NILLQLEEGNPSKLQKIYILDWELCKYGPASLDLGYFLGDCYLISRFQDEQVGTTMRQ AYLQSYARTSKHSINYAKVTAGIAAHIVMWTDFMQWGSEEERINFVKKGVAAFHDA RGNNDNGEITSTLLKESSTA SEQ ID NO: 5 Description: cDNA sequence psiM (P. cyanescens) ATGCATATCAGAAATCCTTACAGAGATGGTGTTGATTATCAAGCTCTAGCTGAAG CATTCCCAGCTCTAAAACCACATGTTACTGTTAATTCTGATAACACAACATCTATC GACTTCGCTGTTCCAGAAGCTCAAAGATTATATACTGCTGCTCTATTGCATAGAG ACTTCGGTCTAACTATTACATTGCCAGAAGATAGATTGTGTCCAACAGTTCCAAA TAGATTAAATTACGTTCTGTGGGTTGAAGATATTCTAAAAGTTACATCTGACGCTC TAGGTCTACCAGATAATAGACAAGTTAAAGGTATTGATATCGGTACAGGTGCTTC TGCTATATATCCAATGTTAGCTTGTTCTAGATTCAAAACATGGTCTATGGTTGCTA CTGAAGTTGATCAAAAATGTATTGATACCGCTAGATTGAATGTTATTGCTAATAA TCTGCAGGAAAGATTGGCTATTATTGCTACATCTGTTGATGGTCCAATTCTAGTTC CATTGTTGCAAGCTAATTCTGACTTCGAATATGACTTCACTATGTGTAATCCACCA TTCTATGATGGTGCTTCTGATATGCAAACATCTGATGCTGCTAAAGGATTCGGATT CGGTGTTAATGCTCCACATACAGGTACAGTTCTAGAAATGGCTACTGAAGGTGGT GAATCTGCATTCGTTGCTCAAATGGTTAGAGAATCTCTAAATCTACAAACTAGAT GTAGATGGTTCACATCTAATCTAGGTAAATTGAAATCCTTGTACGAAATTGTTGG TCTATTAAGAGAACATCAAATCTCTAATTACGCTATTAACGAATACGTTCAAGGT GCTACTAGAAGATATGCTATTGCTTGGTCATTCATTGATGTTAGATTGCCAGATCA TCTATCTAGACCATCTAATCCAGACTTATCTTCTCTATTCTAA SEQ ID NO: 6 Description: amino acid sequence psiM (P. cyanescens) MHIRNPYRDGVDYQALAEAFPALKPHVTVNSDNTTSIDFAVPEAQRLYTAALLHRDF GLTITLPEDRLCPTVPNRLNYVLWVEDILKVTSDALGLPDNRQVKGIDIGTGASAIYP MLACSRFKTWSMVATEVDQKCIDTARLNVIANNLQERLAIIATSVDGPILVPLLQANS DFEYDFTMCNPPFYDGASDMQTSDAAKGFGFGVNAPHTGTVLEMATEGGESAFVAQ MVRESLNLQTRCRWFTSNLGKLKSLYEIVGLLREHQISNYAINEYVQGATRRYAIAW SFIDVRLPDHLSRPSNPDLSSLF SEQ ID NO: 7 Description: cDNA sequence psiH (P. cyanescens) ATGATCGTTCTATTGGTTAGTCTAGTTCTAGCTGGTTGTATATATTATGCTAATGC TAGAAGAGTCAGAAGATCTAGATTGCCACCAGGTCCACCAGGTATTCCATTGCCA TTCATTGGTAATATGTTCGATATGCCATCTGAATCTCCATGGTTGAGATTCTTGCA ATGGGGTAGAGATTATCATACTGATATTCTATACCTGAACGCTGGTGGTACTGAA ATTATTATTCTAAATACCCTGGACGCTATTACAGATCTATTGGAAAAAAGAGGTT CTATGTATTCTGGTAGATTAGAATCTACTATGGTTAATGAACTGATGGGTTGGGA ATTCGATCTAGGATTCATTACTTATGGTGAAAGATGGAGAGAAGAAAGAAGAAT GTTCGCTAAAGAATTCTCTGAAAAAAACATCAGACAGTTCAGACATGCTCAAATT AAAGCTGCTAATCAATTAGTTAGGCAATTGATTAAGACCCCAGATAGATGGTCTC AACATATTAGACATCAAATTGCTGCTATGTCTCTAGATATTGGTTATGGTATTGAT CTAGCTGAAGATGATCCATGGATTGCTGCTACTCAATTGGCTAATGAAGGTCTAG CTGAAGCTTCTGTTCCAGGTTCATTCTGGGTTGATTCATTCCCAGCTCTAAAATAT CTACCATCTTGGTTGCCAGGTGCTGGATTCAAAAGAAAAGCTAAAGTCTGGAAAG AAGGTGCTGATCATATGGTTAATATGCCATATGAAACAATGAAGAAGTTGACTGT TCAAGGTCTAGCTAGACCATCTTATGCTTCTGCTAGATTGCAAGCTATGGACCCTG ATGGTGATCTAGAACATCAAGAACATGTTATTAGAAACACTGCTACTGAAGTTAA TGTTGGTGGTGGTGATACAACTGTTAGTGCTGTTAGTGCATTCATTCTAGCTATGG TTAAATATCCAGAAGTTCAAAGACAAGTTCAAGCTGAATTGGATGCTCTAACATC TAAAGGTGTTGTTCCAAATTATGATGAAGAAGATGATTCTCTACCATATCTAACT GCTTGTGTTAAAGAAATATTCAGATGGAATCAGATCGCTCCATTGGCTATTCCAC ATAGATTAATTAAAGACGATGTCTATAGGGGTTATCTAATTCCAAAAAATGCTCT AGTCTATGCTAATTCTTGGGCTGTTCTAAATGATCCAGAAGAATATCCAAATCCA TCTGAATTCAGACCAGAAAGATATCTATCTTCTGATGGTAAACCAGATCCAACTG TTAGAGATCCAAGAAAAGCTGCATTCGGTTATGGTAGAAGAAATTGTCCAGGTAT TCATCTAGCTCAATCTACAGTCTGGATTGCTGGTGCTACTCTATTATCTGTCTTCA ATATTGAAAGGCCAGTTGATGGTAATGGTAAACCAATTGATATTCCAGCTACATT CACAACTGGATTCTTCAGACATCCAGAACCATTCCAATGTAGATTCGTTCCAAGA ACTCAAGAAATTCTAAAATCTGTTAGCGGTTAA SEQ ID NO: 8 Description: amino acid sequence psiH (P. cyanescens) MIVLLVSLVLAGCIYYANARRVRRSRLPPGPPGIPLPFIGNMFDMPSESPWLRFLQWG RDYHTDILYLNAGGTEIIILNTLDAITDLLEKRGSMYSGRLESTMVNELMGWEFDLGF ITYGERWREERRMFAKEFSEKNIRQFRHAQIKAANQLVRQLIKTPDRWSQHIRHQIAA MSLDIGYGIDLAEDDPWIAATQLANEGLAEASVPGSFWVDSFPALKYLPSWLPGAGF KRKAKVWKEGADHMVNMPYETMKKLTVQGLARPSYASARLQAMDPDGDLEHQEH VIRNTATEVNVGGGDTTVSAVSAFILAMVKYPEVQRQVQAELDALTSKGVVPNYDE EDDSLPYLTACVKEIFRWNQIAPLAIPHRLIKDDVYRGYLIPKNALVYANSWAVLNDP EEYPNPSEFRPERYLSSDGKPDPTVRDPRKAAFGYGRRNCPGIHLAQSTVWIAGATLL SVFNIERPVDGNGKPIDIPATFTTGFFRHPEPFQCRFVPRTQEILKSVSG SEQ ID NO: 9 Description: cDNA sequence SAH1 (P. pastoris) ATGTCTAACTACAAAGTCGCCGACATTTCACTTGCTGCCTTCGGTAGAAAGGACA TTGAACTCAGTGAGAATGAGATGCCAGGTCTCATTTACATCAGAGAGAAGTACGG ACCTGCCCAACCTTTGAAAGGTGCCAGAATCGCCGGATGTCTGCACATGACTATT CAAACCGCCGTCCTCATTGAGACTTTGGTCGCCTTGGGTGCTGAGGTCACCTGGT CCTCATGTAACATTTTCTCCACCCAGGACCACGCTGCCGCTGCTATTGCTGCTACC GGTGTTCCAGTCTTTGCCTGGAAGGGAGAGACCGAGGAGGAGTACTTGTGGTGTA TCGAGCAACAATTATTTGCCTTCAAGGACAACAAGAAGCTGAACTTGATTTTGGA CGACGGTGGTGATTTGACTTCTTTGGTCCACGAGAAGTACCCTGAAATGTTGGAT GACTGTTTCGGTCTGTCCGAGGAGACCACCACTGGTGTCCACCACTTGTACAAGA TGGTCAAGGATGCTACCTTGAAGGTTCCTGCCATCAACGTCAACGACTCCGTCAC CAAGTCCAAGTTTGACAACTTGTACGGTTGTCGTGAATCTTTGATCGACGGTATC AAGCGTGCCACCGATGTTATGATCGCAGGTAAGGTTGCCGTTGTCGCTGGTTTCG GTGACGTTGGTAAAGGTTGTGCCATGGCTCTTAGAGGTATGGGTGCCAGAGTTAT CATCAGTGAGATTGACCCTATCAACGCTCTGCAAGCTGCTGTTGAAGGTTACCAA GTTGCCCCTCTTGATGACGTTGTCTCCATTGGTCAAATCTTTGTTACCACCACTGG TTGCAGAGACATCATCACCGGTAAGCACTTCGAGCAAATGCCAGAAGATGCCATT GTCTCCAACATTGGTCACTTCGACATTGAGATTGACGTTGCTTGGTTGAAGGCCA ACGCTCAGGACGTCAGCAACATCAAGCCTCAAGTTGACAGATACTTAATGAAGA ATGGTCGTCACGTTATTCTTTTGGCTGACGGTAGATTGGTCAACTTGGGTTGTGCC ACTGGTCACTCTTCTTTCGTCATGTCCTGTTCTTTCTCTAACCAGGTCCTGGCTCAA ATTGCTCTGTTCAAGTCTAACGACAGTGAGTTCAGAAAGCAATTCGTTGAGTTCG AAAAGTCTGGTCCATTCGATGTTGGTGTCCACGTTTTACCAAAAATCTTGGATGA AACTGTTGCCAGATGCCATTTGGCTCACTTAGGTGCTAAGCTGACCAAGTTGTCC AGTGTTCAATCTGAGTACTTAGGTATCCCAGTTGAGGGACCTTTCAAGGTTGATC ACTACCGTTACTAG SEQ ID NO: 10 Description: amino acid sequence SAH1 (P. pastoris) MSNYKVADISLAAFGRKDIELSENEMPGLIYIREKYGPAQPLKGARIAGCLHMTIQTA VLIETLVALGAEVTWSSCNIFSTQDHAAAAIAATGVPVFAWKGETEEEYLWCIEQQL FAFKDNKKLNLILDDGGDLTSLVHEKYPEMLDDCFGLSEETTTGVHHLYKMVKDAT LKVPAINVNDSVTKSKFDNLYGCRESLIDGIKRATDVMIAGKVAVVAGFGDVGKGC AMALRGMGARVIISEIDPINALQAAVEGYQVAPLDDVVSIGQIFVTTTGCRDIITGKHF EQMPEDAIVSNIGHFDIEIDVAWLKANAQDVSNIKPQVDRYLMKNGRHVILLADGRL VNLGCATGHSSFVMSCSFSNQVLAQIALFKSNDSEFRKQFVEFEKSGPFDVGVHVLPK ILDETVARCHLAHLGAKLTKLSSVQSEYLGIPVEGPFKVDHYR

Claims

CLAIMS 1. A method for producing a target alkaloid from a precursor via a metabolic pathway in a recombinant yeast cell, wherein the metabolic pathway comprises a psiH enzyme, a psiK enzyme and a psiM enzyme, the method comprising culturing the recombinant yeast cell in a culture medium comprising the precursor under conditions suitable to allow the precursor to enter the recombinant yeast cell such that the recombinant yeast cell produces the target alkaloid, wherein the precursor is a substrate for the psiH enzyme.
2. The method according to claim 1, wherein the target alkaloid is according to formula (I):
Figure imgf000058_0001
X is N, S, O or C, wherein when X is N, R8b is absent, and wherein when X is S or O, R8a and R8b are absent, Z is C or N, wherein when Z is N, R2 is absent, R4 is -HPO4, or -OH, Y1 is -N+H(CH3)2, or -N+H2 CH3, R5, R6, R7 are independently selected from H, D, -CFH2, -CHF2 or -CF3, R2, R3a, R3b, R3c, R3d, R8a and R8b are independently selected from H or D.
3. The method according to claim 1 or claim 2, wherein the target alkaloid is psilocybin and the precursor is tryptamine.
4. The method according to claim 1 or claim 2, wherein: X is S, Z is C, R2, R3a, R3b, R3c, R3d, R5, R6, and R7, are H, and the precursor is 2-(benzothien-3-yl)ethylamine.
5. The method according to claim 1 or claim 2, wherein: X is O, Z is C, R2, R3a, R3b, R3c, R3d, R5, R6, and R7, are H, and the precursor is 2-(Benzofuran-3-yl)ethylamine.
6. The method according to claim 1 or claim 2, wherein: X is C, Z is C, R2, R3a, R3b, R3c, R3d, R5, R6, R7,R8a and R8b are H, and the precursor is 2-(1H-inden-3-yl)ethanamine.
7. The method according to claim 1 or claim 2, wherein: X is N, Z is N and R2 is absent, and the precursor is 2-(1H-indazol-3-yl)ethanamine hydrochloride.
8. The method according to claim 1 or claim 2, wherein: X is O, Z is N and R2 is absent, R3a, R3b, R3c, R3d, R5, R6, and R7, are H, and the precursor is 1, 2-Benzisoxazole-3-ethanamine.
9. The method according to any preceding claim, comprising adding the precursor to the culture medium, optionally wherein the precursor is added to the culture medium in batches.
10. The method according to any preceding claim, wherein the conditions suitable to allow the precursor to pass into the recombinant host cell is a pH 6-7.8.
11. The method according to claim 10, wherein the pH is 7.5-7.8.
12. The method according to any preceding claim, wherein the culture medium comprises a phosphate source.
13. The method according to claim 12, wherein the phosphate source is sodium hexametaphosphate.
14. The method according to any preceding claim, wherein the recombinant yeast cell is a Pichia, a Sacchromyces or a Yarrowia cell.
15. The method according to claim 14, wherein the Pichia cell is Pichia pastoris cell
16. The method according to claim 14, wherein the Sacchromyces cell is S. cerevisiae cell or S. pombe cell.
17. The method according to claim 14, wherein the Yarrowia cell is Y. lipolytica cell.
18. The method according to any preceding claim, wherein the recombinant yeast cell expresses a plurality of genes comprising a psiH gene, a psiK gene and a psiM gene.
19. The method according to claim 18, wherein the plurality of genes comprises an exogenous cytochrome P450 reductase (CPR) gene and/or an adenosylhomocysteinase (SAH1) gene.
20. The method of claim 19, wherein the plurality of genes comprises more than one adenosylhomocysteinase (SAH1) gene.
21. The method according to any of claims 18 to 20, wherein the recombinant yeast cell comprises a DNA construct comprising one or more of the plurality of genes under the control of one or more constitutive or inducible promoters.
22. The method according to claim 21, wherein the one or more constitutive or inducible promoters is GAP or AOX1.
23. The method according to claim 21, wherein the DNA construct comprises a psiH gene, a psiK gene, a psiM gene, an exogenous CPR gene and an adenosylhomocysteinase gene, each under the control of a GAP promoter.
24. The method according to any preceding claim, wherein the culturing is a fed-batch process.
25. The method according to any preceding claim, wherein the target alkaloid is produced at a rate of at least 1 g/litre of culture every 24 hours, preferably at least 3 g/litre of culture every 24 hours, more preferably at least 5 g/litre of culture every 24 hours.
26. A recombinant yeast cell comprising a psiH gene, a psiK gene, and a psiM gene, wherein the recombinant yeast cell: (i) does not comprise an L-tryptophan decarboxylase gene; and/or (ii) comprises an exogenous CPR gene and an adenosylhomocysteinase (SAH1) gene.
27. The recombinant yeast cell according to claim 26, wherein at least one of the psiH gene, the psiK gene, and the psiM gene is under the control of a constitutive promoter or an inducible promoter.
28. The recombinant yeast cell according to claim 27, wherein the psiH gene, the psiK gene, the psiM gene, the exogenous CPR gene and the adenosylhomocysteinase (SAH1) gene are each under the control of a constitutive promoter or an inducible promoter.
29. The recombinant yeast cell of claim 27 or claim 28, wherein the constitutive promoter is GAP or the inducible promoter is AOX1.
30. The recombinant yeast cell of claim 29, wherein the promoter is GAP.
31. The recombinant yeast cell according to any of claims 26 to 30, wherein the recombinant yeast cell is a Pichia, a Sacchromyces or a Yarrowia cell.
32. The recombinant yeast cell according to claim 31, wherein the Pichia cell is Pichia pastoris cell.
33. The recombinant yeast cell according to claim 31, wherein the Sacchromyces cell is S. cerevisiae cell or S. pombe cell.
34. The recombinant yeast cell according to claim 31, wherein the Yarrowia cell is Y. lipolytica cell.
35. A compound according to Formula (I), or a pharmaceutically acceptable salt thereof,
Figure imgf000062_0001
wherein: X is S, O, C, or N, wherein when X is S or O, R8a and R8b are absent, and wherein when X is N, R8b is absent, Z is C or N, wherein when Z is N, R2 is absent, and wherein when X is N, Z is N, R4 is -HPO4, or -OH, Y1 is -N+H(CH3)2, or -N+H2 CH3, R5, R6, R7 are independently selected from H, D, -CFH2, -CHF2 or -CF3, R2, R3a, R3b, R3c, R3d, R8a and R8b are independently selected from H, or D.
36. The compound according to claim 35, wherein X is S or O and R8a and R8b are absent.
37. The compound according to claim 36, wherein X is S and Z is C.
38. The compound according to claim 36, wherein X is O and Z is C.
39. The compound according to claim 36, wherein X is O and Z is N.
40. The compound according to claim 35, wherein X is C and Z is C.
41. The compound according to claim 35, wherein X is N and Z is N.
42. The compound according to any of claims 35 to 41, wherein R4 is -HPO4.
43. The compound according to any of claims 35 to 41, wherein R4 is -OH.
44. The compound according to any of claims 35 to 43, wherein R3a, R3b, R3c, R3d are H.
45. The compound according to any of claims 35 to 44, wherein at least one of R2, R5, R6 and R7 is H.
46. The compound according to claim 45, wherein R2, R5, R6 and R7 are all H.
47. The compound according to any of claims 35 to 46, wherein X is C or N and at least one of R8a and R8b is H, or wherein X is C and R8a and R8b are both H.
48. The compound according to any of claims 35 to 47, wherein Y1 is -N+H(CH3)2.
49. A pharmaceutical composition comprising the compound as defined in any of claims 35 to 48.
50. A pharmaceutical composition according to claim 49, further comprising a pharmaceutically acceptable additive and/or excipient, and/or wherein the compound is in the form of a pharmaceutically acceptable salt.
51. A compound according to any of claims 35 to 48, or a pharmaceutical composition according to claim 49 or claim 50, for use as a medicament.
52. A compound according to any of claims 35 to 48, or a pharmaceutical composition according to claim 49 or claim 50, for use in treating or preventing a mental health condition or disorder.
53. The compound or pharmaceutical composition for use according to claim 52, for use in treating or preventing a mental health condition or disorder by modulating activity of a 5- hydroxytryptamine receptor.
54. The compound or pharmaceutical composition for use according to claim 52 or claim 53, wherein the mental health condition or disorder is selected from anxiety disorder, bipolar disorder, dementia, ADHD, schizophrenia, OCD, autism, PTSD, addiction/substance abuse, post-partum depression, suicidal thoughts, and depression.
55. The compound or pharmaceutical composition for use according to claim 54, wherein the depression is treatment-resistant depression or major depressive disorder.
56. A compound according to any of claims 35 to 48, or a pharmaceutical composition according to claim 49 or claim 50, for use in treating a neurodegenerative disease or disorder, or a brain injury.
57. The compound or pharmaceutical composition for use according to claim 56, for use in treating a neurodegenerative disease or disorder, or a brain injury, by increasing neuroplasticity.
58. The compound or pharmaceutical composition for use according to claim 56 or claim 57, wherein the neurodegenerative disease is Alzheimer’s disease or Parkinson’s disease, or wherein the brain injury is caused by a stroke.
59. A compound according to any of claims 35 to 48, or a pharmaceutical composition according to claim 49 or claim 50, for use in treating or preventing an estrogen-related disease.
60. The compound or pharmaceutical composition for use according to claim 59, wherein the estrogen-related disease is cancer, infertility caused by ovulatory dysfunction, and post- menopause osteoporosis.
61. The compound or pharmaceutical composition for use according to claim 60, wherein the cancer is estrogen receptor positive breast cancer.
62. A compound according to any of claims 35 to 48, or a pharmaceutical composition according to claim 49 or claim 50, for use in the prevention or treatment of inflammation.
63. A compound according to any of claims 35 to 48, or a pharmaceutical composition according to claim 49 or claim 50, for use in treating or preventing an autoimmune disease, optionally wherein the use is as an anti-inflammatory agent.
64. The compound or pharmaceutical composition for use according to claim 63, wherein the autoimmune disease is rheumatoid arthritis, multiple sclerosis, Crohn’s disease, psoriasis, psoriatic arthritis, or type 1 diabetes.
65. A yeast cell culture produced by the method of any of claims 1 to 25 comprising the yeast cell of any of claims 26 to 34 and the target alkaloid.
66. A medicinal formulation comprising yeast cells comprising a target alkaloid and at least one excipient, wherein the yeast cells comprise yeast cells of any of claims 26 to 34 comprising the target alkaloid.
67. The yeast cell culture of claims 65 or the medicinal formulation according to claim 66, wherein the target alkaloid is as defined in any of claims 35 to 48.
68. A yeast cell extract produced by contacting recombinant yeast cell containing a target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell, and centrifuging the extraction mixture to form the yeast cell extract that is the supernatant comprising the target alkaloid, wherein the recombinant yeast cell containing the target alkaloid is produced by the method of any of claims 1 to 25, and wherein the target alkaloid is as defined in any of claims 35 to 48.
69. A method for extracting a produced target alkaloid comprising an amine group and an - HPO4 group from a recombinant yeast cell, the method comprising: (i) contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell; (ii) centrifuging the extraction mixture to form a supernatant comprising the target alkaloid; (iii) ensuring the supernatant has a pH between 10 and 14 and has a low ionic strength solution such that the charge of N in the amine group is neutral; (iv) applying the supernatant to an anion exchange column; and (v) using a hydroxyl ion gradient to elute at least one fraction comprising the target alkaloid.
70. The method according to claim 69, wherein in (i) the recombinant yeast cell is comprised in a whole cell broth and wherein (iii) comprises buffer exchange.
71. The method according to claim 70, wherein buffer exchange comprises nanofiltration.
72. The method according to claim 69, comprising separating the recombinant yeast cell from a culture medium and washing the recombinant yeast cells prior to (i).
73. The method according to any of claims 69 to 72, wherein in (i) the solution comprises 25 to 35% (v/v) acetonitrile.
74. The method according to any of claims 69 to 73, wherein in (iii) the low ionic strength solution is a solution containing 0.05mM to 50 mM ions wherein the ions have a lower selectivity towards the positively charged groups of the anion exchange column than the -HPO4 group.
75. The method according to any of claims 69 to 74, wherein the at least one fraction is two or more fractions and the method comprises combining the two or more fractions, diluting the combined fractions and reapplying to the anion exchange column, and repeating (v).
76. The method according to any of claims 69 to 75, wherein the target alkaloid is according to formula (I) of claim 2.
77. The method according to any one of claims 1 to 25, further comprising extracting the target alkaloid from the recombinant yeast cell using the method for extracting according to any one of claims 69 to 76.
78. A method for increasing methylation capacity of a psiM enzyme in a recombinant yeast cell, the method comprising increasing expression of an adenosylhomocysteinase (SAH1) enzyme in the recombinant yeast cell so as to increase the capacity of the recombinant yeast cell to hydrolyse S-adenosyl-l-homocysteine.
79. The method of claim 78, wherein expression is increased by transforming the yeast cell with one or more copies of an SAH1 gene encoding the SAH1 enzyme; and/or increasing transcription from an SAH1 gene encoding the SAH1 enzyme using a promoter or an enhancer.
80. The method of claim 78 or claim 79, wherein the method is used as part of a method for producing a target alkaloid from a precursor via a metabolic pathway in the recombinant yeast cell, wherein the metabolic pathway comprises a psiH enzyme, a psiK enzyme, a psiM enzyme, and optionally a psiD enzyme.
81. The method of claim 80, wherein the target alkaloid is according to formula I in claim 2.
82. The method according to claim 81, wherein the target alkaloid is psilocybin.
83. The method according to any of claims 80 to 82, wherein the method for producing the target alkaloid is according to any of claims 1 to 25.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021052989A1 (en) 2019-09-16 2021-03-25 Danmarks Tekniske Universitet Yeast cells and methods for production of tryptophan derivatives
US11441164B2 (en) 2019-11-15 2022-09-13 Cb Therapeutics, Inc. Biosynthetic production of psilocybin and related intermediates in recombinant organisms

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI129102B (en) * 2018-03-19 2021-07-15 Teknologian Tutkimuskeskus Vtt Oy Heterologous production of psilocybin
AU2021337466A1 (en) * 2020-09-01 2023-03-30 Enveric Biosciences Canada Inc. Halogenated psilocybin derivatives and methods of using
KR20230157342A (en) * 2021-02-12 2023-11-16 인베릭 바이오사이언시스 캐나다 인크. Multisubstituted psilocybin derivatives and methods of use
WO2022248635A2 (en) * 2021-05-27 2022-12-01 Octarine Bio Aps Methods for producing tryptamine derivatives.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021052989A1 (en) 2019-09-16 2021-03-25 Danmarks Tekniske Universitet Yeast cells and methods for production of tryptophan derivatives
US11441164B2 (en) 2019-11-15 2022-09-13 Cb Therapeutics, Inc. Biosynthetic production of psilocybin and related intermediates in recombinant organisms

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ANDERSEN ET AL., ACTA PSYCHIATR. SCAND., vol. 143, no. 2, February 2021 (2021-02-01), pages 101 - 118
BAI ET AL., EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, vol. 221, 2021, pages 113543
FLANAGANNICHOLS: "Psychedelics as anti-inflammatory agents", INT. REV. PSYCHIATRY, 2018
FRICKE ET AL., ANGEWANDTE CHEMIE, vol. 56, no. 40, 25 September 2017 (2017-09-25), pages 12352 - 12355
KOZLOWSKA ET AL., JOURNAL OF NEUROCHEMISTRY, vol. 162, 2022, pages 89 - 108
KÜBERL ET AL.: "High-quality genome sequence of Pichia pastoris CDS7435", JOURNAL OF BIOTECHNOLOGY, vol. 154, no. 4, 20 July 2011 (2011-07-20), pages 312 - 320
MILNE ET AL., METABOLIC ENGINEERING, vol. 60, 2020, pages 25 - 36
MOLINER ET AL., NATURE NEUROSCIENCE, vol. 26, June 2023 (2023-06-01), pages 1032 - 1041
SIEGMANN ET AL., JAMA PSYCHIATRY, vol. 75, no. 6, 1 June 2018 (2018-06-01), pages 577 - 584
STRUMILANOBILE ET AL., PHARMACEUTICALS, vol. 14, 2021, pages 1213
THOMPSONSZABO, IMMUNOLOGY LETTERS, vol. 228, 2020, pages 45 - 54

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