WO2023081833A1 - Metabolic engineering methods for the production of psilocybin and intermediates or side products - Google Patents

Metabolic engineering methods for the production of psilocybin and intermediates or side products Download PDF

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WO2023081833A1
WO2023081833A1 PCT/US2022/079314 US2022079314W WO2023081833A1 WO 2023081833 A1 WO2023081833 A1 WO 2023081833A1 US 2022079314 W US2022079314 W US 2022079314W WO 2023081833 A1 WO2023081833 A1 WO 2023081833A1
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gene
mutant
sequence
prokaryotic cell
psilocybin
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PCT/US2022/079314
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French (fr)
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John Andrew Jones
Nicholas KAPLAN
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Miami University
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Definitions

  • the general inventive concepts relate to the field of medical therapeutics and more particularly to metabolic engineering methods for the production of psilocybin and intermediates or side products.
  • Psilocybin (4-phosphoryloxy-N, N -dimethyltryptamine) has gained atention in pharmaceutical markets as a result of recent clinical studies. The efficacy of psilocybin has been demonstrated for the treatment of anxiety in terminal cancer patients and alleviating the symptoms of post-traumatic stress disorder (PTSD). Most recently, the FDA has approved the first Phase lib clinical trial for the use of psilocybin as a treatment for depression that is not well controlled with currently available interventions such as antidepressants and cognitive behavioral therapies.
  • Psilocybin was first purified from the Psilocybe mexicana mushroom by the Swiss chemist, Albert Hoffmann, in 1958. The first reports of the complete chemical synthesis of psilocybin were published in 1959; however, large-scale synthesis methods were not developed until the early 2000 ’s by Shirota and colleagues at the National Institute of Sciences in Tokyo. Despite significant improvements over early synthetic routes, current methods remain tedious and costly, involving numerous intermediate separation and purification steps resulting in an overall yield of 49% from 4-hydroxyindole, incurring an estimated cost of $2 USD per milligram for pharmaceutical-grade psilocybin.
  • a method for the production of psilocybin or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression
  • each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell, wherein the host cell is optionally cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, betaine, and combinations thereof.
  • the side product of psilocybin is aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamonium (4-OH-TMT).
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of psiD, psiK, psiM, acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • an expression vector comprising a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof. Also provided is a transfection kit comprising an expression vector as described herein.
  • SUBSTITUTE SHEET (RULE 26)
  • an expression vector comprising a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • a transfection kit comprising an expression vector as described herein.
  • FIG. 1 shows methionine and activated methyl cycle biosynthetic pathways including repressors. Dashed arrows represent a multi-step process abbreviated for simplicity, writing on an arrow represents an enzymatic step, T shapes represent repression, and SahH represents a heterologous step not found in E. coli.
  • FIG. 2 shows a biochemical pathway for psilocybin production with fed substrate, 4- OH indole.
  • FIG. 3 shows a biochemical pathway for psilocybin production de novo.
  • FIG. 7 illustrates layout of mutant libraries.
  • FIG. 8 shows psilocybin production of the mutant library with the luxS-mtn accessory genes for both the 5 g/L methionine and 0 g/L methionine conditions.
  • FIG. 9 shows psilocybin production of the mutant library with the sahH accessory gene for both the 5 g/L methionine and 0 g/L methionine conditions.
  • FIG. 10 shows psilocybin production of the mutant library with the metK accessory gene for both the 5 g/L methionine and 0 g/L methionine conditions.
  • FIG. 11 shows psilocybin production of the mutant library with the metA*-cysE* accessory genes for both the 5 g/L methionine and 0 g/L methionine conditions.
  • FIG. 12 shows PCR validation of 14 top mutants. Expected sizes are 489 bp for luxS- mtn constructs, 499bp for metK constructs, and 559bp for sahH constructs.
  • FIG. 13 shows Validation of Top Performing Mutants.
  • FIG. 14 shows Psilocybin titer as a function of time for select mutants (with methionine supplementation) and the psilocybin production strain (PPS) in both methionine- supplemented and non-supplemented conditions.
  • FIG. 15 shows psilocybin production of a mutant library with BHMT accessory gene under betaine supplementation at 50 mM.
  • a cell means one cell or more than one cell.
  • Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or “consisting essentially of’ such features, and vice-versa.
  • prokaryotic host cell means a prokaryotic cell that is susceptible to transformation, transfection, transduction, or the like, with a nucleic acid construct or expression vector comprising a polynucleotide.
  • prokaryotic host cell encompasses any progeny that is not identical due to mutations that occur during replication.
  • the term “recombinant cell” or “recombinant host” means a cell or host cell that has been genetically modified or altered to comprise a nucleic acid sequence that is not native to the cell or host cell. In some embodiments the genetic modification
  • SUBSTITUTE SHEET (RULE 26) comprises integrating the polynucleotide in the genome of the host cell.
  • the polynucleotide is exogenous in the host cell.
  • intermediate of psilocybin means an intermediate in the production or biosynthesis of psilocybin, e.g., norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine.
  • side product of psilocybin means a side product in the production or biosynthesis of psilocybin, e.g., aeruginascin, psilocin, norpsilocin, or 4- hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT) .
  • Methods for the production of psilocybin or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell, wherein the host cell is optionally cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, betaine, and combinations thereof.
  • the method further comprises contacting the prokaryotic cell with an expression vector comprising a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • the cysE is a feedback inhibition-resistant variant.
  • the metA is a feedback inhibition-resistant variant.
  • the prokaryotic host cell comprises a genomic knockout of metJ. In some embodiments, the prokaryotic host cell comprises a genomic knockout of speD.
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number KY984100.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is selected from the group consisting of
  • Escherichia coli Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces
  • SUBSTITUTE SHEET (RULE 26) coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • any intermediate or side product of psilocybin may be produced by any of the methods described herein.
  • the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT).
  • the intermediate of psilocybin is norbaeocystin, baeocystin, 4- hydroxytryptophan, or 4-hydroxytryptamine.
  • the side product of psilocybin is aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4- OH-TMT).
  • the supplement does not comprise methionine.
  • the supplement is fed continuously to the host cell.
  • the host cell is grown in an actively growing culture
  • the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of psiD, psiK, psiM, acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • the cysE is a feedback inhibition-resistant variant.
  • the metA is a feedback inhibition-resistant variant.
  • the prokaryotic host cell comprises a genomic knockout of metJ. In some embodiments, the prokaryotic host cell comprises a genomic knockout of speD.
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1, or a sequence having at least 60%, at
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number KY984100.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the acs gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the acs is encoded by a nucleotide sequence comprising SEQ ID NO: 40, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metA gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metA is encoded by a nucleotide sequence comprising SEQ ID NO: 24, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the cysE gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or a sequence having at least 60%, at least 70%, at least
  • SUBSTITUTE SHEET (RULE 26) 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the cysE is encoded by a nucleotide sequence comprising SEQ ID NO: 26, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the sahH gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the sahH is encoded by a nucleotide sequence comprising SEQ ID NO: 28, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metK is encoded by a nucleotide sequence comprising SEQ ID NO: 30, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the luxS gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the luxS is encoded by a nucleotide sequence comprising SEQ ID NO: 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • SUBSTITUTE SHEET (RULE 26) least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the SAM2 gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the SAM2 is encoded by a nucleotide sequence comprising SEQ ID NO: 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the BHMT gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 39 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the BHMT is encoded by a nucleotide sequence comprising SEQ ID NO: 38, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the prokaryotic cell comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof, all under control of a single promoter in operon configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9
  • SUBSTITUTE SHEET (RULE 26) mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the accessory gene(s) and the psilocybin production gene(s) are in independent operons.
  • the prokaryotic cell comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
  • all accessory genes are under control of a single promoter in operon configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • an expression vector comprising a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.
  • the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1, or a sequence having at least 60%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising S
  • SUBSTITUTE SHEET (RULE 26) 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number KY984100.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the expression vector comprises a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the expression vector comprises a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
  • the promoter is selected from the
  • SUBSTITUTE SHEET (RULE 26) group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof, all under control of a single promoter in operon configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the accessory gene(s) and the psilocybin production gene(s) are in independent operons.
  • the prokaryotic cell comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
  • all accessory genes are under control of a single promoter in operon configuration.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • an expression vector comprising a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
  • the acs gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the acs is encoded by a nucleotide sequence
  • SUBSTITUTE SHEET (RULE 26) comprising SEQ ID NO: 40, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metA gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metA is encoded by a nucleotide sequence comprising SEQ ID NO: 24, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the cysE gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the cysE is encoded by a nucleotide sequence comprising SEQ ID NO: 26, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the sahH gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the sahH is encoded by a nucleotide sequence comprising SEQ ID NO: 28, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the metK is encoded by a nucleotide sequence comprising SEQ ID NO: 30, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the luxS gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the luxS is encoded by a nucleotide sequence comprising SEQ ID NO: 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the mtn gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the mtn is encoded by a nucleotide sequence comprising SEQ ID NO: 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the SAM2 gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the SAM2 is encoded by a nucleotide sequence comprising SEQ ID NO: 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the BHMT gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 39 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the BHMT is encoded by a nucleotide sequence comprising SEQ ID NO: 38, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • kits comprising an expression vector as described herein.
  • a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes.
  • container means such as, e.g., vials or test tubes.
  • Each of such container means comprises components or a mixture of components needed to perform a transfection.
  • kits may include, for example, one or more components selected from vectors, cells, reagents, lipid-aggregate forming compounds, transfection enhancers, or biologically active molecules.
  • compositions and methods for genetic modification of cells to aid in the production of psilocybin and intermediates or side products according to the general inventive concepts.
  • E. coll DH5a was used to propagate all plasmids, while BL21 starTM (DE3) was used as the host for all chemical production experiments. Plasmid transformations were completed using standard electro and chemical competency protocols as specified. Unless noted otherwise, Andrew's Magic Media (AMM) [35] was used for both overnight growth and production media, while Luria Broth (LB) was used for plasmid propagation during cloning. The antibiotics ampicillin (80pg/mL) for cells containing the pETM6 plasmid and kanamycin (50pg/mL) for the BL21 starTM(DE3) AMetJ::KanR strain were added at their respective concentrations to the culture media.
  • Standard screening was performed in 2 mL working volume cultures in 48-well plates at 37 °C. AMM supplemented with serine (5 g/L), 4-hydroxyindole (350 mg/L), and appropriate antibiotics were used unless otherwise noted. Overnight cultures were grown from either an agar plate or freezer stock culture in AMM with appropriate antibiotics and supplements (5 g/L serine) for 14 h in a shaking 37 °C incubator. Each mutant was screened in two different conditions, with methionine supplementation (5 g/L) and without methionine supplementation. Lor each mutant, induction with 1 mM isopropyl-[3-D-l-
  • SUBSTITUTE SHEET (RULE 26) thiogalactopyranoside (IPTG) occurred 4 h after inoculation. Cultures were then sampled 30 h post inoculation and subjected to HPLC analysis as described below.
  • the bioreactors were inoculated at 2% v/v to an initial OD600 of approximately 0.09.
  • the bioreactors were initially filled with AMM media (1.5 L) supplemented with 150 mg/L 4-hydroxyindole, 5 g/L serine, and 5 g/L methionine (except where noted).
  • Temperature was held constant at 37 °C with a heat jacket and recirculating cooling water, pH was automatically controlled at 6.5 with the addition of 10 M KOH, and dissolved oxygen (DO) was maintained at 30% of saturation through agitation cascade control (250-1000 rpm). Full oxygen saturation was defined under the conditions of 37 °C, pH 7.0, 250 rpm agitation, and 1.5 1pm of standard air.
  • the zero-oxygen set point was achieved by nitrogen purge. Samples were collected periodically for measurement of OD600 and metabolite analysis. The bioreactors were induced with 1 mM IPTG 4 h post inoculation. Once the initial 20 g/L of glucose was reduced to approximately 5 g/L, as identified through HPLC analysis, feed streams of 50% glucose were fed at a mass flow rate ranging from 400 mg/L/hr to 1600 mg/L/hr. Beginning 12 h post inoculation, a continuous supply of 4-hydroxyindole was supplied by external syringe pump to the bioreactor.
  • the feed rate of 4-hydroxyindole was manually varied from 0 to 40 mg/L/hr according to the observed buildup of the pathway intermediates 4-hydroxytryptophan and 4-hydroxyindole.
  • SUBSTITUTE SHEET (RULE 26) immediately analyzed via HPLC on an approximate 30-min delay and were used as feedback into the feeding strategy described above.
  • Native accessory genes (metK, luxS, mtn) were amplified from the E. coli genome using primers shown in Table 1 with the addition of flanking restriction enzyme cut sites (Ndel, Xhol) for insertion into the pETM6 plasmid.
  • SUBSTITUTE SHEET (RULE 26) [0109] After amplification of the native genes or synthetic DNA, amplicons were subjected to restriction enzyme digest with At/el and Xhol. The accessory genes were then gel extracted and ligated to a pETM6-SDM2x plasmid backbone also digested with At/el and Xhol.
  • the pETM6-SDM2x- (Accessory Gene) plasmid was digested with Xma J I and Sall, gel extracted, and ligated with the pETM6 -xx 5 backbone to produce the pETM6-xx 5 -(Accessory Gene) libraries.
  • the ligation product was transformed into E. coll DH5oc and plated on agar plates with ampicillin. After 16 hours, all colonies on agar plate were scraped and plasmid purified via mini-prep to attain the pETM6-xx 5 -(Accessory Gene) plasmid library.
  • pETM6- HlO-PsiD-PsiK-PsiM psilocybin production strain
  • pETM6-xx5 -mCherry stock discussed above was also digested with Xbal and Apal and gel extracted to purify the pETM6-xx5 backbone.
  • These extractions were ligated and transformed into E. coll DH5 ⁇ plated on agar plates with ampicillin. After 16 hours, all colonies on agar plate were scraped and plasmid purified via mini-prep to attain the pETM6-xx 5 -PsiD-PsiK-PsiM plasmid library.
  • the pETM6 -xx 5 -(Accessory Gene) plasmid was digested with Nhel and Apal and the backbone was gel extracted.
  • the pETM6-xx 5 -PsiD-PsiK-PsiM library was digested with Xma J I and Apal and the xx 5 -PsiD-PsiK-PsiM operon was extracted for insertion into the pETM6-xx 5 -(Accessory Gene) backbone. These extractions were ligated and transformed into E. coll DH5oc and plated on agar plates with ampicillin.
  • the purified plasmid DNA was transformed into electrocompetent BL21 starTM (DE3). This transformation was plated on agar plates with ampicillin. To validate performance of the mutants, a swab of colonies was inoculated into 48-well plates in methionine supplemented and non-supplemented conditions. These validation experiments were performed in triplicate, including the psilocybin production strain as a positive control. Mutants demonstrating reproducibility were selected for scale-up testing in bench-top bioreactors.
  • Glucose and acetate concentrations in the growth medium were also monitored throughout the course of the experiment. According to a study published in 2002, addition of acetate to the growth medium has an inhibitory effect on cellular growth, which can be relieved by addition of methionine to the growth medium 17 .
  • the authors demonstrate that treating cells with acetate at 8 mM causes both a buildup of the toxic intermediate, homocysteine, and a reduction in methionine pools that is limiting to growth. It has been shown that homocysteine inhibits branched-chain amino acid biosynthesis 23 . They suggest that acetate may serve to inhibit an enzymatic step in the AMC, causing a bottleneck that can be relieved by supplementing methionine. This information is valuable as both the growth of E. colt and production of psilocybin are dependent on the AMC functioning efficiently.
  • a plot showing glucose and acetate concentrations as a function of time is shown in Figure 5.
  • SUBSTITUTE SHEET (RULE 26) of 0 g/L in the +Met replicates between 24 and 36 hours, corresponding to a drop in acetate levels characteristic of the acetate switch. It is important to note that although glucose levels were maintained at exhaustion, glucose was still being fed at a low rate throughout the course of the experiments. In the fermentations without methionine supplemented, acetate levels continued to increase throughout the experiment as glucose levels did not become limiting.
  • Table 2 Summary of gene targets for manipulation, including gene source (native/non- native/engineered), intended manipulation, intended result, and reference.
  • SUBSTITUTE SHEET (RULE 26) psilocybin production is growth-dependent in the current system, maximizing growth is of primary concern. Moreover, the biochemical pathway for psilocybin production is directly dependent on SAM, the primary product of the activated methyl cycle. It is hypothesized that metabolic engineering techniques to reduce acetate buildup will increase growth and psilocybin production in this strain.
  • Efforts to reduce acetate buildup in fermentations are well described in the literature by several different approaches including overexpression of endogenous genes that elevate pyruvate flux into the TCA cycle 26 , expression of exogenous genes that convert pyruvate into waste species less toxic than acetate 27 , and overexpression of an endogenous gene that increases utilization of acetate to produce acetyl -coA 25 .
  • the approach that focused on increasing acetate consumption as a carbon source is of particular interest, as it recycles waste carbon resulting in higher carbon yields. This manipulation was achieved by overexpressing acetyl-coA synthetase (ACS) on a vector.
  • ACS acetyl-coA synthetase
  • Acetyl-coA is an important molecule that supplies the acetyl group to the TCA cycle, and is generally utilized by E. coli in carbohydrate, lipid, and protein metabolism. It was demonstrated that strains with ACS overexpression had reduced levels of acetate accumulation, higher growth rates, and higher glucose consumption rates than the wild-type 25 . Due to the advantages discussed above in growth and acetate consumption, ACS, encoded by the native gene acs. is a target for overexpression in this strain. This gene can be attained by genomic PCR amplification as it is native to E. coli. The primers used for amplification are stated in the methods section of Lin et al 25 .
  • SAH the toxic chemical species produced by SAM utilization
  • luxS and mtn are regenerated into SAM naturally in E. coli by gene products of luxS and mtn.
  • SAM production and utilization quantities are intended to be amplified by metabolic engineering strategies discussed in this work, it is expected that natural degradation pathways will also need to be amplified to minimize SAH accumulation throughout the fermentation.
  • overexpression of luxS and mtn have been demonstrated to increase production of the heterologous chemical product, vanillate, by 25% 12 . Due to the production enhancement seen in this comparable study, luxS and mtn are overexpressed together.
  • the luxS gene product produces Al -2, which has a significant and wide-spread effect on gene regulation, including activity associated with the activated methyl cycle 11 . It has not been investigated whether global gene regulation is
  • SUBSTITUTE SHEET (RULE 26) affected by overexpression of this gene. If overexpression of /ux S has unintended global effects on gene transcription, significant contribution to metabolic burden could be expected.
  • sahH As shown in Figure 1, the gene product of sahH can catalyze degradation of SAH to homocysteine in one step rather than two.
  • sahH is a gene found in eukaryotes and prokaryotes that do not contain luxS.
  • overexpression of this non-native gene in E. coli could improve AMC efficiency by offering an alternative pathway for toxic SAH degradation.
  • This hypothesis was investigated by Kunjapur et al 12 , and expression of this heterologous gene from .S', cerevisiae was found to decrease production of the chemical product, vanillate.
  • this result was not rigorously investigated and enzyme activity was not established, suggesting the potential of sahH expression should not be discounted in all scenarios.
  • heterologous gene sahH was expressed in the same dual-operon library discussed above, and mutants were screened for psilocybin production with and without methionine supplementation. Several sahH mutants outperformed the psilocybin production strain, establishing a successful rebalancing of the psilocybin biosynthesis pathway with the accessory gene. The top five mutants in the methionine -supplemented condition were isolated for sequencing and validation. See Figure 9.
  • luxS Like the overexpression of luxS, overexpression of sahH may interfere with quorum sensing, due to a reduction of SRH levels caused by the direct conversion of SAH to HCY.
  • luxS produces DPD as a byproduct of the conversion of SRH to HCY.
  • DPD is spontaneously converted to AI-2, which is the signaling molecule of quorum sensing in E. coli. Reduced intracellular pools of SRH may have implications with quorum sensing in this strain.
  • Another method for increasing SAM pools is the overexpression of metK, which catalyzes the conversion of methionine to SAM.
  • metK catalyzes the conversion of methionine to SAM.
  • the overexpression of this accessory gene theoretically improves AMC efficiency by ‘pulling’ intermediates through the cycle with SAM as the desired product. It can also help to convert methionine supplemented in the media to SAM, dependent on the rate of transport of methionine into the cell. In a methionine-supplemented condition, this manipulation could increase methionine flux into the activated methyl cycle by readily utilizing intracellular methionine.
  • the methionine transporter in E. colt utilizes active transport (requiring ATP) and is inhibited allosterically by intracellular methionine. If the overexpression of metK maintains low intracellular methionine concentrations, the rate of methionine transport into the cell may increase, thereby increasing methionine flux into the AMC.
  • metK was placed into the dual operon structure for random promoter optimization. These mutants were screened for psilocybin production in methionine-supplemented and non-supplemented conditions. The screening results of the metK accessory gene are shown in Figure 10. It was observed that several mutants outperformed the psilocybin production strain in this screen. These results suggest that the psilocybin bioproduction platform was successfully rebalanced, with the potential for added functionality due to the presence of the accessory gene. The five mutants which resulted in the highest psilocybin titers with methionine supplementation were isolated for sequencing and validation.
  • the observed detriment to psilocybin production may be due to the increased carbon flux into the AMC, without any metabolic engineering to increase the regenerative capacity of SAM. This may have resulted in an accumulation of SAH and subsequent inhibition of the PsiM methyltransferase.
  • BHMT betaine-homocystine methyltransferase
  • the 5 mutant promoters used in this study are shown below in Table 4, each differing in 5 nucleotide bases which are highlighted for emphasis. These promoters are arranged in order of decreasing end-point expression strength, as characterized by Jones et al [22] .
  • FIG. 14 shows psilocybin titer for each mutant as a function of time, with dashed lines representing the data recorded for the psilocybin production strain from Figure 4. Growth was consistent for each reactor as measured by ODeoo (data not shown), and differences in growth behavior were not found to be statistically significant (p>0.05).
  • mutants KD10 and SH2 performed similarly to the psilocybin production strain until the end of the fermentation. Production was closely linked to growth,
  • Strain LD2 (with methionine supplementation) performed similarly to the psilocybin production strain in the non-supplemented condition. This may indicate that this particular mutant introduced a SAM-deficiency, rather than the intended purpose of increasing SAM pools. The poor performance of this mutant may not be due to the presence of luxS and mtn, but the particular pathway balancing between the accessory and psilocybin operons. To investigate this hypothesis, other mutants containing the luxS-mtn accessory operon should be scaled in bioreactor conditions.
  • Asterisk (*) represents genes which have enabled higher titers when expressed together in the literature.
  • Cells labeled ‘A’ represent combinatorial expression that is hypothesized to be generally beneficial.
  • Cells labeled ‘B’ represent combinatorial expression that is hypothesized to be redundant.
  • Table 6 represents a proposed plasmid list for intended overexpressions used in this project. Each of these plasmids is tested in a fermentation run with pETM6-SDM2x-PsiD-PsiK-PsiM, on the wild-type genomic background, LmetJ background, LspeD background, and
  • strains have been identified which result in either improved AMC dynamics or increased psilocybin production, these strains are run in scale-up studies using bench-scale bioreactors to test strains for decreased dependency on methionine. If the bioreactor experiments result in a reduced dependency on methionine supplementation, it can be assumed that AMC efficiency has been increased, and a promoter library for the psilocybin pathway will be tested on this genetic background to optimize psilocybin titers as described by Adams et al. Additionally, promoter libraries can be constructed for the overexpression plasmids used in that strain to further optimize metabolic efficiency and psilocybin production.

Abstract

Provided is a method for the production of psilocybin or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell, wherein the host cell is optionally cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, betaine, and combinations thereof. In some embodiments, the expression vector further comprises a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof. In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.

Description

METABOLIC ENGINEERING METHODS FOR THE PRODUCTION OF PSILOCYBIN AND INTERMEDIATES OR SIDE PRODUCTS
FIELD
[0001] The general inventive concepts relate to the field of medical therapeutics and more particularly to metabolic engineering methods for the production of psilocybin and intermediates or side products.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] The instant application is entitled to priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63/263,614, filed November 5, 2021, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0003] The contents of the electronic sequence listing (315691-00043.xml; Size: 85,725 bytes; and Date of Creation: November 4, 2022) is herein incorporated by reference in its entirety.
BACKGROUND
[0004] Because of its potential for treatment for a number of anxiety and mental-health related conditions, interest in psilocybin is significant. However, due to roadblocks in routing methods of obtaining drug targets (synthesis and/or extraction from a known biological source), large amounts are not currently available.
[0005] As proof-of-principle metabolic engineering projects are pushed toward commercial application, significant roadblocks exist in the scale-up process including low product yields, reduced growth rates, and biochemical instability caused by the introduction of heterologous pathways. Many of these problems can be attributed to the complex, highly evolved regulatory networks found in endogenous metabolism. As carbon flux is redirected toward a heterologous product in a host organism, the naturally evolved metabolic balance is disrupted, likely demanding further manipulation to realize full growth and production potential. With the goal of increasing titers of various high-value chemical products, metabolic engineering techniques are being investigated.
SUBSTITUTE SHEET (RULE 26) [0006] Psilocybin (4-phosphoryloxy-N, N -dimethyltryptamine) has gained atention in pharmaceutical markets as a result of recent clinical studies. The efficacy of psilocybin has been demonstrated for the treatment of anxiety in terminal cancer patients and alleviating the symptoms of post-traumatic stress disorder (PTSD). Most recently, the FDA has approved the first Phase lib clinical trial for the use of psilocybin as a treatment for depression that is not well controlled with currently available interventions such as antidepressants and cognitive behavioral therapies.
[0007] Psilocybin was first purified from the Psilocybe mexicana mushroom by the Swiss chemist, Albert Hoffmann, in 1958. The first reports of the complete chemical synthesis of psilocybin were published in 1959; however, large-scale synthesis methods were not developed until the early 2000 ’s by Shirota and colleagues at the National Institute of Sciences in Tokyo. Despite significant improvements over early synthetic routes, current methods remain tedious and costly, involving numerous intermediate separation and purification steps resulting in an overall yield of 49% from 4-hydroxyindole, incurring an estimated cost of $2 USD per milligram for pharmaceutical-grade psilocybin.
[0008] Much of the interest in psilocybin is due to its biosynthetic precursors — norbaeocystin and baeocystin. These compounds have structural similarity to the neurotransmiter serotonin and sparked the interest of researchers who were curious to understand the mechanism behind their hallucinogenic properties. After being named a Schedule I compound in the US with implementation of the Controlled Substance Act of 1970, research efforts involving psilocybin were abandoned for other less regulated bioactive molecules; however, experts in the field have suggested a reclassification to schedule IV would be appropriate if a psilocybin-containing medicine were to be approved in the future.
[0009] Clinical trials with psilocybin as a medication for individuals struggling with treatment-resistant depression are ongoing.
[0010] There remains a need for improved methods for the production of psilocybin and intermediates or side products thereof through metabolic engineering.
SUMMARY
[0011] Provided is a method for the production of psilocybin or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression
SUBSTITUTE SHEET (RULE 26) vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell, wherein the host cell is optionally cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, betaine, and combinations thereof.
[0012] In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus , and Streptomyces venezuelae.
[0013] In some embodiments, the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamonium (4-OH-TMT). In some embodiments the intermediate of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, or 4- hydroxytryptamine. In some embodiments, the side product of psilocybin is aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamonium (4-OH-TMT).
[0014] Also provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of psiD, psiK, psiM, acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
[0015] Also provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
[0016] Also provided is an expression vector comprising a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof. Also provided is a transfection kit comprising an expression vector as described herein.
SUBSTITUTE SHEET (RULE 26) [0017] Also provided is an expression vector comprising a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof. Also provided is a transfection kit comprising an expression vector as described herein.
DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows methionine and activated methyl cycle biosynthetic pathways including repressors. Dashed arrows represent a multi-step process abbreviated for simplicity, writing on an arrow represents an enzymatic step, T shapes represent repression, and SahH represents a heterologous step not found in E. coli.
[0019] FIG. 2 shows a biochemical pathway for psilocybin production with fed substrate, 4- OH indole.
[0020] FIG. 3 shows a biochemical pathway for psilocybin production de novo.
[0021] FIG. 4 shows a progress curve showing ODeoo and psilocybin titer in 2L bench-scale bioreactors with methionine supplemented (+Met) or no methionine supplemented (-Met). Error bars represent standard deviation of duplicate experiments (N=2).
[0022] FIG. 5 is a graph that shows Acetate (A) and glucose (G) concentrations in the growth medium as a function of time for bioreactors which were supplemented with methionine (+Met) and bioreactors which were not supplemented with methionine (-Met). Error bars represent standard deviation of duplicate experiments (N=2).
[0023] FIGs. 6A-6B show psilocybin intermediate buildup in psilocybin production strain bioreactors as a function of time. Error bars represent standard deviation of duplicate experiments (N=2).
[0024] FIG. 7 illustrates layout of mutant libraries.
[0025] FIG. 8 shows psilocybin production of the mutant library with the luxS-mtn accessory genes for both the 5 g/L methionine and 0 g/L methionine conditions. The solid gray bar represents the psilocybin production strain without accessory genes (N =4) and the gradient bars represent mutants (N=l). Error bars signify standard error of the mean.
[0026] FIG. 9 shows psilocybin production of the mutant library with the sahH accessory gene for both the 5 g/L methionine and 0 g/L methionine conditions. The solid gray bar
SUBSTITUTE SHEET (RULE 26) represents the psilocybin production strain without accessory genes (N =4) and the gradient bars represent mutants (N=l). Error bars signify standard error of the mean.
[0027] FIG. 10 shows psilocybin production of the mutant library with the metK accessory gene for both the 5 g/L methionine and 0 g/L methionine conditions. The solid gray bar represents the psilocybin production strain without accessory genes (N =4) and the gradient bars represent mutants (N=l). Error bars signify standard error of the mean.
[0028] FIG. 11 shows psilocybin production of the mutant library with the metA*-cysE* accessory genes for both the 5 g/L methionine and 0 g/L methionine conditions. The solid gray bar represents the psilocybin production strain without accessory genes (N =4) and the gradient bars represent mutants (N=l). Error bars signify standard error of the mean.
[0029] FIG. 12 shows PCR validation of 14 top mutants. Expected sizes are 489 bp for luxS- mtn constructs, 499bp for metK constructs, and 559bp for sahH constructs.
[0030] FIG. 13 shows Validation of Top Performing Mutants. The first letter of each mutant name signifies the accessory gene (S = sahH, L = luxS-mtn, K = metK), and the following digits signify the position of the mutant in the 96-well glycerol freezer stock. Error bars signify standard error of the mean for replicates (N=3).
[0031] FIG. 14 shows Psilocybin titer as a function of time for select mutants (with methionine supplementation) and the psilocybin production strain (PPS) in both methionine- supplemented and non-supplemented conditions. The first letter of each mutant name signifies the accessory gene (S = sahH, L = luxS-mtn, K = metK), and the following digits signify the position of the mutant in the 96-well glycerol freezer stock. Error bars represent standard deviation of duplicates (N=2).
[0032] FIG. 15 shows psilocybin production of a mutant library with BHMT accessory gene under betaine supplementation at 50 mM. The solid gray bar represents the psilocybin production strain without accessory genes (N =4) and the black bars represent mutants (N=l). Error bars signify standard deviation from the mean.
DETAILED DESCRIPTION
[0033] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments
SUBSTITUTE SHEET (RULE 26) thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.
[0034] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0035] The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a cell” means one cell or more than one cell.
[0036] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0037] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or “consisting essentially of’ such features, and vice-versa.
[0038] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range in explicitly recited.
[0039] As used herein, the term “prokaryotic host cell” means a prokaryotic cell that is susceptible to transformation, transfection, transduction, or the like, with a nucleic acid construct or expression vector comprising a polynucleotide. The term “prokaryotic host cell” encompasses any progeny that is not identical due to mutations that occur during replication.
[0040] As used herein, the term “recombinant cell” or “recombinant host” means a cell or host cell that has been genetically modified or altered to comprise a nucleic acid sequence that is not native to the cell or host cell. In some embodiments the genetic modification
SUBSTITUTE SHEET (RULE 26) comprises integrating the polynucleotide in the genome of the host cell. In further embodiments the polynucleotide is exogenous in the host cell.
[0041] As used herein, the term “intermediate” of psilocybin means an intermediate in the production or biosynthesis of psilocybin, e.g., norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine.
[0042] As used herein, the term “side product” of psilocybin means a side product in the production or biosynthesis of psilocybin, e.g., aeruginascin, psilocin, norpsilocin, or 4- hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT) .
[0043] The materials, compositions, and methods described herein are intended to be used to provide novel routes for the production of psilocybin and intermediates or side products.
Methods for the production of psilocybin or an intermediate or a side product thereof [0044] Provided is a method for the production of psilocybin or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell, wherein the host cell is optionally cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, betaine, and combinations thereof.
[0045] In some embodiments, the method further comprises contacting the prokaryotic cell with an expression vector comprising a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof. In further embodiments, the cysE is a feedback inhibition-resistant variant. In further embodiments, the metA is a feedback inhibition-resistant variant.
[0046] In some embodiments, the prokaryotic host cell comprises a genomic knockout of metJ. In some embodiments, the prokaryotic host cell comprises a genomic knockout of speD.
[0047] In some embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
SUBSTITUTE SHEET (RULE 26) sequence identity thereto. In some embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0048] In some embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0049] In some embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0050] In some embodiments, the prokaryotic cell is selected from the group consisting of
Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces
SUBSTITUTE SHEET (RULE 26) coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0051] In some embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0052] In some embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0053] It is envisaged that any intermediate or side product of psilocybin may be produced by any of the methods described herein. In some embodiments, the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT). In some embodiments the intermediate of psilocybin is norbaeocystin, baeocystin, 4- hydroxytryptophan, or 4-hydroxytryptamine. In some embodiments, the side product of psilocybin is aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4- OH-TMT).
[0054] In some embodiments, the supplement does not comprise methionine.
[0055] In some embodiments, the supplement is fed continuously to the host cell.
[0056] In some embodiments, the host cell is grown in an actively growing culture
[0057] In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
SUBSTITUTE SHEET (RULE 26) Recombinant prokaryotic cells for the production of psilocybin or an intermediate or a side product thereof
[0058] Also provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of psiD, psiK, psiM, acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
[0059] Also provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof. In further embodiments, the cysE is a feedback inhibition-resistant variant. In further embodiments, the metA is a feedback inhibition-resistant variant.
[0060] In some embodiments, the prokaryotic host cell comprises a genomic knockout of metJ. In some embodiments, the prokaryotic host cell comprises a genomic knockout of speD.
[0061] In some embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0062] In some embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1, or a sequence having at least 60%, at
SUBSTITUTE SHEET (RULE 26) least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0063] In some embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0064] In some embodiments, the acs gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the acs is encoded by a nucleotide sequence comprising SEQ ID NO: 40, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0065] In some embodiments, the metA gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the metA is encoded by a nucleotide sequence comprising SEQ ID NO: 24, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0066] In some embodiments, the cysE gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or a sequence having at least 60%, at least 70%, at least
SUBSTITUTE SHEET (RULE 26) 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the cysE is encoded by a nucleotide sequence comprising SEQ ID NO: 26, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0067] In some embodiments, the sahH gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the sahH is encoded by a nucleotide sequence comprising SEQ ID NO: 28, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0068] In some embodiments, the metK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the metK is encoded by a nucleotide sequence comprising SEQ ID NO: 30, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0069] In some embodiments, the luxS gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the luxS is encoded by a nucleotide sequence comprising SEQ ID NO: 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0070] In some embodiments, the mtn gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the mtn is encoded by a nucleotide sequence comprising SEQ ID NO: 34, or a sequence having at least 60%, at least 70%, at least 80%, at
SUBSTITUTE SHEET (RULE 26) least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0071] In some embodiments, the SAM2 gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the SAM2 is encoded by a nucleotide sequence comprising SEQ ID NO: 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0072] In some embodiments, the BHMT gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 39 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the BHMT is encoded by a nucleotide sequence comprising SEQ ID NO: 38, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0073] In some embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynehacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0074] In some embodiments, the prokaryotic cell comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0075] In some embodiments, the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof, all under control of a single promoter in operon configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9
SUBSTITUTE SHEET (RULE 26) mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter. In some embodiments, the accessory gene(s) and the psilocybin production gene(s) are in independent operons.
[0076] In some embodiments, the prokaryotic cell comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0077] In some embodiments, the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof. In some embodiments, each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. In some embodiments, all accessory genes are under control of a single promoter in operon configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0078] Any configuration or arrangement of promoters and terminators is envisaged.
Expression vectors
[0079] Also provided is an expression vector comprising a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.
[0080] In some embodiments, the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1, or a sequence having at least 60%, at least
SUBSTITUTE SHEET (RULE 26) 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0081] In some embodiments, the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0082] In some embodiments, the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0083] In some embodiments, the expression vector comprises a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0084] In some embodiments, the expression vector comprises a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. In further embodiments, the promoter is selected from the
SUBSTITUTE SHEET (RULE 26) group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0085] In some embodiments, the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof, all under control of a single promoter in operon configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter. In some embodiments, the accessory gene(s) and the psilocybin production gene(s) are in independent operons.
[0086] In some embodiments, the prokaryotic cell comprises an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0087] In some embodiments, the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof. In some embodiments, each gene is under control of a separate promoter in pseudooperon or monocistronic configuration. In some embodiments, all accessory genes are under control of a single promoter in operon configuration. In further embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0088] Any configuration or arrangement of promoters and terminators is envisaged.
[0089] Also provided is an expression vector comprising a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
[0090] In some embodiments, the acs gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the acs is encoded by a nucleotide sequence
SUBSTITUTE SHEET (RULE 26) comprising SEQ ID NO: 40, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0091] In some embodiments, the metA gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the metA is encoded by a nucleotide sequence comprising SEQ ID NO: 24, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0092] In some embodiments, the cysE gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the cysE is encoded by a nucleotide sequence comprising SEQ ID NO: 26, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0093] In some embodiments, the sahH gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the sahH is encoded by a nucleotide sequence comprising SEQ ID NO: 28, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0094] In some embodiments, the metK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the metK is encoded by a nucleotide sequence comprising SEQ ID NO: 30, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
SUBSTITUTE SHEET (RULE 26) [0095] In some embodiments, the luxS gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the luxS is encoded by a nucleotide sequence comprising SEQ ID NO: 32, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0096] In some embodiments, the mtn gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the mtn is encoded by a nucleotide sequence comprising SEQ ID NO: 34, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0097] In some embodiments, the SAM2 gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the SAM2 is encoded by a nucleotide sequence comprising SEQ ID NO: 36, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0098] In some embodiments, the BHMT gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 39 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the BHMT is encoded by a nucleotide sequence comprising SEQ ID NO: 38, or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0099] Any configuration or arrangement of promoters and terminators is envisaged.
SUBSTITUTE SHEET (RULE 26) Kits
[0100] Provided is a transfection kit comprising an expression vector as described herein. Such a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes. Each of such container means comprises components or a mixture of components needed to perform a transfection. Such kits may include, for example, one or more components selected from vectors, cells, reagents, lipid-aggregate forming compounds, transfection enhancers, or biologically active molecules.
EXAMPLES
[0101] The following examples describe various compositions and methods for genetic modification of cells to aid in the production of psilocybin and intermediates or side products, according to the general inventive concepts.
Methods
Bacterial Strains, Vectors, and Media
[0102] E. coll DH5a was used to propagate all plasmids, while BL21 star™ (DE3) was used as the host for all chemical production experiments. Plasmid transformations were completed using standard electro and chemical competency protocols as specified. Unless noted otherwise, Andrew's Magic Media (AMM) [35] was used for both overnight growth and production media, while Luria Broth (LB) was used for plasmid propagation during cloning. The antibiotics ampicillin (80pg/mL) for cells containing the pETM6 plasmid and kanamycin (50pg/mL) for the BL21 star™(DE3) AMetJ::KanR strain were added at their respective concentrations to the culture media.
Standard Screening Conditions
[0103] Standard screening was performed in 2 mL working volume cultures in 48-well plates at 37 °C. AMM supplemented with serine (5 g/L), 4-hydroxyindole (350 mg/L), and appropriate antibiotics were used unless otherwise noted. Overnight cultures were grown from either an agar plate or freezer stock culture in AMM with appropriate antibiotics and supplements (5 g/L serine) for 14 h in a shaking 37 °C incubator. Each mutant was screened in two different conditions, with methionine supplementation (5 g/L) and without methionine supplementation. Lor each mutant, induction with 1 mM isopropyl-[3-D-l-
SUBSTITUTE SHEET (RULE 26) thiogalactopyranoside (IPTG) occurred 4 h after inoculation. Cultures were then sampled 30 h post inoculation and subjected to HPLC analysis as described below.
Analytical Methods
[0104] Analytical methods for quantification of psilocybin and glucose concentrations in fermentation runs were performed as described by Adams et al[19]. Additionally, norbaeocystin and baeocystin standard curves were constructed using in-house prep-HPLC purified product.
Fermentation Conditions
[0105] Scale-up studies were performed in duplicate i n Eppendorf BioFlo120 bioreactors with 1.5 L working volume. The cylindrical vessels were mixed by a direct drive shaft containing two Rushton-type impellers positioned equidistance under the liquid surface. The overnight cultures of the psilocybin production strain used to inoculate the bioreactors was grown for 12 h at 37 °C in AMM supplemented with serine (5 g/L), appropriate antibiotics, and either supplemented with methionine (5 g/L) or not supplemented methionine (0 g/L), matching respective fermentation conditions. The overnight cultures were inoculated from a glycerol stock stored at -80 °C. The bioreactors were inoculated at 2% v/v to an initial OD600 of approximately 0.09. The bioreactors were initially filled with AMM media (1.5 L) supplemented with 150 mg/L 4-hydroxyindole, 5 g/L serine, and 5 g/L methionine (except where noted). Temperature was held constant at 37 °C with a heat jacket and recirculating cooling water, pH was automatically controlled at 6.5 with the addition of 10 M KOH, and dissolved oxygen (DO) was maintained at 30% of saturation through agitation cascade control (250-1000 rpm). Full oxygen saturation was defined under the conditions of 37 °C, pH 7.0, 250 rpm agitation, and 1.5 1pm of standard air. The zero-oxygen set point was achieved by nitrogen purge. Samples were collected periodically for measurement of OD600 and metabolite analysis. The bioreactors were induced with 1 mM IPTG 4 h post inoculation. Once the initial 20 g/L of glucose was reduced to approximately 5 g/L, as identified through HPLC analysis, feed streams of 50% glucose were fed at a mass flow rate ranging from 400 mg/L/hr to 1600 mg/L/hr. Beginning 12 h post inoculation, a continuous supply of 4-hydroxyindole was supplied by external syringe pump to the bioreactor. The feed rate of 4-hydroxyindole was manually varied from 0 to 40 mg/L/hr according to the observed buildup of the pathway intermediates 4-hydroxytryptophan and 4-hydroxyindole. The concentration of psilocybin, all intermediate compounds, glucose, and acetate w ere
SUBSTITUTE SHEET (RULE 26) immediately analyzed via HPLC on an approximate 30-min delay and were used as feedback into the feeding strategy described above.
[0106] Library Construction
[0107] Native accessory genes (metK, luxS, mtn) were amplified from the E. coli genome using primers shown in Table 1 with the addition of flanking restriction enzyme cut sites (Ndel, Xhol) for insertion into the pETM6 plasmid.
[0108] The nucleotide sequence of heterologous sahH, and engineered metA* and cysE* are published by Kunjapur et al12 and ordered from Azenta Life Sciences as linear, double stranded DNA with flanking Ndel and Xhol restriction enzyme cut sites for subsequent insertion into the pETM6 plasmid.
Table 1. Primer list. Ndel and Xhol recognition sequences are underlined.
Figure imgf000023_0001
SUBSTITUTE SHEET (RULE 26) [0109] After amplification of the native genes or synthetic DNA, amplicons were subjected to restriction enzyme digest with At/el and Xhol. The accessory genes were then gel extracted and ligated to a pETM6-SDM2x plasmid backbone also digested with At/el and Xhol.
[0110] To produce the pETM6-xx5-(Accessory Gene) libraries, equimolar amounts of the plasmids pETM6-T7-m Cherry, pETM6-C4-mCherry, pETM6-H10-mCherry, pETM6-H9- mCherry, and pETM6-G6-mCherry were combined and digested with Xbal and Sall to extract the pETM6-xx5 backbone and remove the mCherry gene. The pETM6-SDM2x- (Accessory Gene) plasmid was digested with Xma J I and Sall, gel extracted, and ligated with the pETM6 -xx5 backbone to produce the pETM6-xx5-(Accessory Gene) libraries. The ligation product was transformed into E. coll DH5oc and plated on agar plates with ampicillin. After 16 hours, all colonies on agar plate were scraped and plasmid purified via mini-prep to attain the pETM6-xx5-(Accessory Gene) plasmid library.
[0111] To attain the random mutant library for the psilocybin biosynthesis pathway, pETM6- HlO-PsiD-PsiK-PsiM (psilocybin production strain) was digested with Xbal wA Apa\ and gel extracted to isolate the PsiD-PsiK-PsiM pathway. The pETM6-xx5 -mCherry stock discussed above was also digested with Xbal and Apal and gel extracted to purify the pETM6-xx5 backbone. These extractions were ligated and transformed into E. coll DH5α plated on agar plates with ampicillin. After 16 hours, all colonies on agar plate were scraped and plasmid purified via mini-prep to attain the pETM6-xx5 -PsiD-PsiK-PsiM plasmid library.
[0112] Finally, the pETM6 -xx5-(Accessory Gene) plasmid was digested with Nhel and Apal and the backbone was gel extracted. The pETM6-xx5 -PsiD-PsiK-PsiM library was digested with Xma J I and Apal and the xx5-PsiD-PsiK-PsiM operon was extracted for insertion into the pETM6-xx5-(Accessory Gene) backbone. These extractions were ligated and transformed into E. coll DH5oc and plated on agar plates with ampicillin. After 16 hours, all colonies on agar plate were scraped and plasmid purified via mini-prep to attain the pETM6-xx5- (Accessory Gene)-xx5 -PsiD-PsiK-PsiM plasmid library. This plasmid mixture was transformed into chemically competent BL21 star™ (DE3) cells and individual colonies were picked and inoculated into 48 well plates for the mutant screening experiments.
Mutant Isolation and Validation
SUBSTITUTE SHEET (RULE 26) [0113] Glycerol stocks maintained at -80 °C were created from the overnight culture for each mutant of the promoter libraries. Following data analysis, the five mutants with the highest psilocybin titer in the methionine -supplemented condition of each library, with the exception of metA*-cysE* libraries, were inoculated into an overnight culture and mini-prepped to isolate the plasmid. Once isolated, the plasmid was transformed into chemically competent E. coli DH5a by heat shock at 42 °C. A single colony was picked from the resulting agar plates and inoculated into an overnight culture. After 16 hours, this overnight was also mini prepped and the resulting plasmid DNA stock was subjected to PCR amplification as a preliminary screen for presence of the accessory operon and Sanger sequencing for sequence verification and promoter characterization.
[0114] After the presence of accessory genes was validated, the purified plasmid DNA was transformed into electrocompetent BL21 star™ (DE3). This transformation was plated on agar plates with ampicillin. To validate performance of the mutants, a swab of colonies was inoculated into 48-well plates in methionine supplemented and non-supplemented conditions. These validation experiments were performed in triplicate, including the psilocybin production strain as a positive control. Mutants demonstrating reproducibility were selected for scale-up testing in bench-top bioreactors.
Example 1: Methionine Supplementation Scale-Up Study
[0115] With the goal of further optimizing the strain described by Adams et al. for both higher psilocybin titers and reduced toxicity of activated methyl cycle intermediates, experiments were designed to further characterize the effect of SAM availability on psilocybin production in the current strain. Without wishing to be bound by theory, it was hypothesized that increasing SAM availability would lead to an increase in psilocybin titers, since SAM is utilized as a methyl donor by PsiM22. It has been shown that the supplementation of methionine, a direct SAM precursor, to the growth medium increases psilocybin production in well plate and shake flask fermentations15, suggesting SAM availability may be limited in this process. However, the effects of methionine supplementation at scale-up had not yet been investigated. With the goal of optimizing a psilocybin producing strain for industrial use, it was of particular interest to investigate the effects of SAM availability at scale-up. Fermentation methods are detailed elsewhere herein.
Impacts of Methionine Supplementation on Growth and Psilocybin Production
SUBSTITUTE SHEET (RULE 26) [0116] To investigate the effect of methionine supplementation at scale-up, four bench-scale bioreactors operating under previously described best conditions were run in parallel, with (5 g/L) and without (0 g/L) methionine supplementation in the growth medium. A progress curve attained from this experiment is shown in Figure 4. Bioreactors with methionine supplementation significantly outperformed bioreactors without methionine supplementation (p=0.003), representing a greater than 2-fold improvement in psilocybin titer. It was also observed that the bioreactors run with methionine supplementation seemed to reach higher optical densities, but this difference was not found to be statistically significant at the 95% confidence interval (p=0.19). The significant difference in psilocybin production between the two conditions motivates the need for further improvements in this strain.
[0117] Although the bioreactors that were supplemented with methionine outperformed the bioreactors without supplementation, it is notable that this deviation did not become significant until after 24 hours of the fermentation, at a titer of around 300 mg/L of psilocybin.
Acetate Accumulation
[0118] Glucose and acetate concentrations in the growth medium were also monitored throughout the course of the experiment. According to a study published in 2002, addition of acetate to the growth medium has an inhibitory effect on cellular growth, which can be relieved by addition of methionine to the growth medium17. The authors demonstrate that treating cells with acetate at 8 mM causes both a buildup of the toxic intermediate, homocysteine, and a reduction in methionine pools that is limiting to growth. It has been shown that homocysteine inhibits branched-chain amino acid biosynthesis23. They suggest that acetate may serve to inhibit an enzymatic step in the AMC, causing a bottleneck that can be relieved by supplementing methionine. This information is valuable as both the growth of E. colt and production of psilocybin are dependent on the AMC functioning efficiently. A plot showing glucose and acetate concentrations as a function of time is shown in Figure 5.
[0119] It can be seen in the replicates with methionine supplementation that acetate levels decrease after around 24 hours of growth. This behavior can be explained by the so called “acetate switch”24. This phenomenon occurs as acetate-producing carbon sources, like glucose, are depleted in the growth medium. In order to survive, the cells begin to uptake and metabolize acetate. As shown in Figure 5, glucose levels reach and remain at a concentration
SUBSTITUTE SHEET (RULE 26) of 0 g/L in the +Met replicates between 24 and 36 hours, corresponding to a drop in acetate levels characteristic of the acetate switch. It is important to note that although glucose levels were maintained at exhaustion, glucose was still being fed at a low rate throughout the course of the experiments. In the fermentations without methionine supplemented, acetate levels continued to increase throughout the experiment as glucose levels did not become limiting. In an identical bioreactor experiment resulting in a similar, 2-fold improvement in psilocybin production with methionine supplementation, neither condition ran out of glucose and differences in acetate buildup were not significant between methionine supplemented and non-supplemented conditions, making the presumption that varying acetate accumulation caused the differences seen in psilocybin production between the two conditions very unlikely.
[0120] However, it is probable that acetate buildup is generally limiting growth and psilocybin production in this strain. In the bioreactor replicates in which no methionine was added, acetate levels climbed to a final growth medium concentration of 6.5 g/L (approximately 110 mM), well beyond the 8 mM acetate concentration established to inhibit growth by Roe et al17. In the replicates in which methionine was supplemented and acetate was consumed, acetate levels remained higher than 1.2 g/L (approximately 20 mM) throughout the course of the experiment, which is higher than the concentration utilized to study inhibitory effects of acetate in E. coli. With the goal of increasing methionine pools and maximizing growth potential of this strain, rational engineering attempts should be made, besides running at glucose exhaustion, to reduce acetate buildup during fermentation. A metabolic engineering approach to reduce acetate buildup is proposed elsewhere herein.
Psilocybin Pathway Intermediates
[0121] In addition to glucose and acetate, concentrations of psilocybin pathway intermediates were monitored throughout the course of the bioreactor experiments. These results are shown in Figures 6A-6B. As described elsewhere herein, PsiM catalyzes two methylation reactions including: 1) methylation of norbaeocystin yielding baeocystin and 2) methylation of baeocystin yielding psilocybin. As SAM is a substrate for PsiM it was hypothesized that in experiments without methionine supplementation (SAM-limited), co-substrates norbaeocystin and baeocystin levels would build up in the growth medium. Indeed, in the replicates without methionine supplementation, there was a buildup of 178 mg/L of norbaeocystin at the end of the fermentation, whereas there were only trace amounts of
SUBSTITUTE SHEET (RULE 26) norbaeocystin present in the replicates with methionine supplemented. Additionally, there was over twice as much baeocystin present at the end of the fermentation in SAM-limited replicates when compared with replicates with methionine supplementation. The respective concentrations of baeocystin are not known, as standards for construction of a calibration curve are not commercially available. However, these findings further elucidate the SAM- limiting behavior observed without methionine supplementation and the necessity for manipulation of endogenous metabolism in the creation of a robust, industrial strain.
Example 2: Targeted Genes
Gene targets which have been selected a priori will be discussed to inform strategic manipulations that may be performed in the construction of a more robust psilocybin production strain. These manipulations will be characterized individually, and then applied in a combinatorial approach to optimize psilocybin production. A table summarizing the following discussion is shown below in Table 2.
Table 2. Summary of gene targets for manipulation, including gene source (native/non- native/engineered), intended manipulation, intended result, and reference.
Figure imgf000028_0001
Overexpression of acetyl-coA synthetase (ACS) for Reduced Acetate Accumulation
[0122] As discussed elsewhere herein, acetate buildup during fermentation is known to have inhibitory effects on the activated methyl cycle, causing growth defects in E. coli. Because
SUBSTITUTE SHEET (RULE 26) psilocybin production is growth-dependent in the current system, maximizing growth is of primary concern. Moreover, the biochemical pathway for psilocybin production is directly dependent on SAM, the primary product of the activated methyl cycle. It is hypothesized that metabolic engineering techniques to reduce acetate buildup will increase growth and psilocybin production in this strain. Efforts to reduce acetate buildup in fermentations are well described in the literature by several different approaches including overexpression of endogenous genes that elevate pyruvate flux into the TCA cycle26, expression of exogenous genes that convert pyruvate into waste species less toxic than acetate27, and overexpression of an endogenous gene that increases utilization of acetate to produce acetyl -coA25. The approach that focused on increasing acetate consumption as a carbon source is of particular interest, as it recycles waste carbon resulting in higher carbon yields. This manipulation was achieved by overexpressing acetyl-coA synthetase (ACS) on a vector. Acetyl-coA is an important molecule that supplies the acetyl group to the TCA cycle, and is generally utilized by E. coli in carbohydrate, lipid, and protein metabolism. It was demonstrated that strains with ACS overexpression had reduced levels of acetate accumulation, higher growth rates, and higher glucose consumption rates than the wild-type25. Due to the advantages discussed above in growth and acetate consumption, ACS, encoded by the native gene acs. is a target for overexpression in this strain. This gene can be attained by genomic PCR amplification as it is native to E. coli. The primers used for amplification are stated in the methods section of Lin et al25.
Overexpression of luxS and mtn for Improved SAH Degradation
[0123] SAH, the toxic chemical species produced by SAM utilization, is regenerated into SAM naturally in E. coli by gene products of luxS and mtn. However, as SAM production and utilization quantities are intended to be amplified by metabolic engineering strategies discussed in this work, it is expected that natural degradation pathways will also need to be amplified to minimize SAH accumulation throughout the fermentation. Moreover, overexpression of luxS and mtn have been demonstrated to increase production of the heterologous chemical product, vanillate, by 25%12. Due to the production enhancement seen in this comparable study, luxS and mtn are overexpressed together.
[0124] As discussed elsewhere herein, the luxS gene product produces Al -2, which has a significant and wide-spread effect on gene regulation, including activity associated with the activated methyl cycle11. It has not been investigated whether global gene regulation is
SUBSTITUTE SHEET (RULE 26) affected by overexpression of this gene. If overexpression of /ux S has unintended global effects on gene transcription, significant contribution to metabolic burden could be expected.
Overexpression of Heterologous sahH for Improved SAH Degradation
[0125] As shown in Figure 1, the gene product of sahH can catalyze degradation of SAH to homocysteine in one step rather than two. sahH is a gene found in eukaryotes and prokaryotes that do not contain luxS. However, it is hypothesized that overexpression of this non-native gene in E. coli could improve AMC efficiency by offering an alternative pathway for toxic SAH degradation. This hypothesis was investigated by Kunjapur et al12, and expression of this heterologous gene from .S', cerevisiae was found to decrease production of the chemical product, vanillate. However, it was noted that this result was not rigorously investigated and enzyme activity was not established, suggesting the potential of sahH expression should not be discounted in all scenarios.
[0126] The heterologous gene sahH was expressed in the same dual-operon library discussed above, and mutants were screened for psilocybin production with and without methionine supplementation. Several sahH mutants outperformed the psilocybin production strain, establishing a successful rebalancing of the psilocybin biosynthesis pathway with the accessory gene. The top five mutants in the methionine -supplemented condition were isolated for sequencing and validation. See Figure 9.
[0127] It is notable that the number of mutants which demonstrated appreciable psilocybin titers is small compared to the results observed in other libraries. This behavior may be due to the dependence of SahH on NAD+ as a cofactor. It is plausible that high expression levels of sahH result in a redox imbalance which impedes psilocybin production.
[0128] Like the overexpression of luxS, overexpression of sahH may interfere with quorum sensing, due to a reduction of SRH levels caused by the direct conversion of SAH to HCY. As discussed elsewhere herein, luxS produces DPD as a byproduct of the conversion of SRH to HCY. DPD is spontaneously converted to AI-2, which is the signaling molecule of quorum sensing in E. coli. Reduced intracellular pools of SRH may have implications with quorum sensing in this strain.
SUBSTITUTE SHEET (RULE 26) Overexpression of metK
[0129] Another method for increasing SAM pools is the overexpression of metK, which catalyzes the conversion of methionine to SAM. The overexpression of this accessory gene theoretically improves AMC efficiency by ‘pulling’ intermediates through the cycle with SAM as the desired product. It can also help to convert methionine supplemented in the media to SAM, dependent on the rate of transport of methionine into the cell. In a methionine-supplemented condition, this manipulation could increase methionine flux into the activated methyl cycle by readily utilizing intracellular methionine. The methionine transporter in E. colt utilizes active transport (requiring ATP) and is inhibited allosterically by intracellular methionine. If the overexpression of metK maintains low intracellular methionine concentrations, the rate of methionine transport into the cell may increase, thereby increasing methionine flux into the AMC.
[0130] Like the accessory genes described above, metK was placed into the dual operon structure for random promoter optimization. These mutants were screened for psilocybin production in methionine-supplemented and non-supplemented conditions. The screening results of the metK accessory gene are shown in Figure 10. It was observed that several mutants outperformed the psilocybin production strain in this screen. These results suggest that the psilocybin bioproduction platform was successfully rebalanced, with the potential for added functionality due to the presence of the accessory gene. The five mutants which resulted in the highest psilocybin titers with methionine supplementation were isolated for sequencing and validation.
[0131] With the addition of methionine to the growth medium, a wide range of titers were observed. However, without the addition of methionine, there seemed to be two distinct titer levels around 50 and 100 mg/L. One explanation for this behavior is that overexpression of metK causes an exhaustion of methionine at the intracellular concentrations present in endogenous metabolism, and no further improvement in psilocybin production is seen, regardless of the expression strength of the psilocybin pathway. However, when methionine is supplemented, a wider range of titers are observed, dependent upon both the expression strength of metK and the psilocybin biosynthesis pathway. Analysis of the promoter pairings of each mutant in this library and dynamic profiling of AMC metabolites could enable an
SUBSTITUTE SHEET (RULE 26) exploration of this hypothesis, however exploration of this mechanism is beyond the scope of this work.
Overexpression of Feedback-Resistant variants, cysE* and met A *
[0132] As discussed elsewhere herein, flux into the activated methyl cycle is controlled by feedback inhibition of CysE and MetA. At high intracellular concentrations, methionine and SAM inhibit CysE and MetA, and thus reduce carbon flux into the AMC9’12. As SAM levels have proven to be limiting for maximum psilocybin production in our strain, it is hypothesized that circumventing feedback inhibition will increase flux of precursors homoserine and cysteine into the AMC and result in elevated SAM production. It is demonstrated by Kunjapur et al that overexpressing feedback-resistant variants of cysE and metA, known as cysE* and metA*, resulted in a 33% increase in vanillate titers when coupled with gene inactivation of the MetJ repressor. The DNA sequences of these mutant enzymes are published by Kunjapur et al12. In this work, metA* and cysE* were expressed on a plasmid in both the BL21 star™(DE3) strain and the BL21 star™(DE3) AmetJ::KanR strains.
[0133] The screening results for the mutant library with metA* and cysE* accessory genes in the BL21 star™(DE3) wild-type strain are shown in Figure 11. The addition of these accessory genes appeared to be detrimental to psilocybin production in both the methionine supplemented and non-supplemented conditions. All mutants of this library demonstrated psilocybin titers below 50 mg/L, whereas the psilocybin production strain resulted in an average titer greater than 250 mg/L.
[0134] Without wishing to be bound by theory, the observed detriment to psilocybin production may be due to the increased carbon flux into the AMC, without any metabolic engineering to increase the regenerative capacity of SAM. This may have resulted in an accumulation of SAH and subsequent inhibition of the PsiM methyltransferase.
Overexpressing these feedback inhibition-resistant enzymes in conjunction with accessory genes which improve regenerative capacity of the AMC is recommended.
Amei.J Knockout and Overexpression of metA *-cysE*
[0135] Since nearly every gene responsible for AMC enzymes is under negative transcriptional control of the gene product of metJ, it is hypothesized that inactivation of metJ will allow higher flux through the AMC, resulting in increased SAM availability for growth
SUBSTITUTE SHEET (RULE 26) and product formation. When combined with the overexpression of cysE* and met A *. the deletion of metJ was shown to increase vanillate titers by 33%12 motivating this deletion for improved psilocybin production.
[0136] The BL21 star™(DE3) Amet.I strain was created using the Datsenko-Wanner method of chromosomal inactivation [33], Preliminary testing by the applicants showed the Amet.I strain background hinders titers of the psilocybin production strain without accessory genes. To further explore this phenomenon, the metA*-cysE* library was transformed into the BL21 star™(DE3) Emei.J ::KanR strain in which the met J gene encoding the transcriptional repressor for AMC genes was knocked out. Trace amounts of psilocybin were detected via mass spectroscopy, suggesting the psilocybin biosynthesis pathway was active, but measurable psilocybin concentrations were not detected by UV-VIS for any of the mutants.
[0137] Due to the low titers observed when the same metA*-cysE* library was transformed into the wild-type the BL21 star™(DE3) strain, the poor performance seen in the Amet.I strain could be attributed to the accessory operon. Without any manipulations to directly improve SAM regeneration, the further removal of highly-evolved metabolic regulation strategies may have caused a detrimental accumulation of toxic intermediates. As discussed elsewhere herein, other downregulation techniques like CRISPRi may allow tunable repression of met J while leaving some regulatory features in place.
Silencing of speD for Reduced Decarboxylation of SAM
[0138] It was shown by Bao et al. that sRNA mediated repression of speD, a native SAM- utilizing enzyme, resulted in a 172% increase in psilocybin production when compared to the wild-type21. When a combinatorial approach was applied, combining this speD repression with repression of acrF and tynA, even greater results were demonstrated. acrF encodes a transporter that removes indole from the cell, and tynA encodes a competing tryptophan utilization enzyme. In this thesis, repression of acrF and tynA is not desired, as removing a transporter which likely exports 4-OH indole from the cell could result in increased toxicity effects. Additionally, tynA is a competing pathway for tryptophan consumption, but in the 4- OH indole -dependent psilocybin production platform, tryptophan is not a substrate. With the long-term goal of improving de novo synthesis of psilocybin, this discussion involving tynA may be revisited.
SUBSTITUTE SHEET (RULE 26) BHMT and SAM2
[0139] The gene encoding betaine-homocystine methyltransferase (BHMT) was cloned into the dual operon structure for random promoter optimization in an analogous fashion to that of other genes in this study. BHMT catalyzes the conversion of homocysteine into methionine through the consumption of the methyl donor, betaine, resulting in the formation of a dimethylglycine byproduct. This action bypasses the folate cycle and activated methyl cycle (AMC) members MetH and MetE and is hypothesized to reduce the burden associated with the regeneration of L-5-methyltetrahydrofolate (5-MTHF). The screening results with BHMT are shown in Figure 15. Here, the top performing mutants encoding BHMT variants (black bars) are equivalent to the positive control, psilocybin production strain (gray bar, second from the left). This indicates that the optimization strategy used was able to achieve the rebalancing of the pathway which is necessary after the combination of the BHMT gene and the psilocybin pathway on a single vector.
[0140] The screening of 140 transcriptionally varied mutants produced many productive strains, however BHMT overexpression alone was not sufficient to meaningfully enhance production, even after promoter optimization. Without wishing to be bound by theory, this suggests that the homocysteine -to-methionine catalytic step is not the sole rate limiting step for the AMC. Taken together with the other single accessory gene overexpression studies, this suggests that there is not a single overexpression capable of significant enhancement in AMC activity. Looking forward, combinations of accessory genes capable of enhancing full AMC flux may be necessary to alleviate bottlenecks encountered in the biosynthesis of methylated products in microbial hosts.
Example 3: Mutant Sequencing and Validation
[0141] Top-producing mutants from each library screen experiment (with the exception of metA*-cysE* accessory gene libraries) were chosen and isolated for promoter characterization and fermentation behavior validation in triplicate. PCR amplification was used to probe the presence of the intended accessory gene before mutants were submitted for Sanger sequencing. The resulting 2% agarose gel is shown in Figure 12. A band of appropriate size was observed for each of the 14 mutants tested, suggesting the presence of the accessory gene in this plasmid. The reverse primers bind inside the accessory gene sequence and thus amplification would not be observed in the absence of the accessory gene.
SUBSTITUTE SHEET (RULE 26) It was observed that the bands seemed to run ~50bp larger than expected. The plasmid DNA used for PCR amplification was submitted for Sanger sequencing for further analysis of the amplicon sizes seen on the gel.
[0142] After the preliminary PCR amplification to check for the presence of accessory genes in each mutant, Sanger sequencing was used to confirm the accessory gene sequences and characterize the promoters on each operon. Table 3 reveals the promoter characterization of the accessory gene operon and the psilocybin biosynthesis pathway operon from Sanger sequencing results.
Table 3. Promoter characterization for the two operons for select mutants. Sequencing reactions failed for several mutants, and these spaces are labeled “unk”.
Figure imgf000035_0001
[0143] The 5 mutant promoters used in this study are shown below in Table 4, each differing in 5 nucleotide bases which are highlighted for emphasis. These promoters are arranged in order of decreasing end-point expression strength, as characterized by Jones et al [22] .
SUBSTITUTE SHEET (RULE 26) Table 4. Mutant promoters of the xx5 library and their respective sequences. Varying sequences are underlined.
Figure imgf000036_0001
[0144] As shown in Table 4, the top performing mutants tended to have a stronger promoter controlling the psilocybin operon, with a weaker promoter controlling the accessory gene operon. This suggests the most productive mutants have a higher expression level of the psilocybin biosynthesis pathway than the accessory gene(s).
[0145] As described elsewhere herein, the plasmid isolated from select mutants was retransformed into E. coli BL21 star™ (DE3) for validation of psilocybin production. The results of this validation experiment are shown in Figure 13. This data further suggests the ability of several mutants to overcome metabolic burden associated with the addition of accessory genes and prompts the investigation of enhanced biochemical functionality at scale-up.
Example 4: Bioreactor Scale-up
[0146] As screening results for several top-performing mutants demonstrated that the metabolic burden associated with the addition of accessory genes could be overcome by pathway balancing via promoter optimization, it was of primary interest to next investigate whether these accessory genes provided improved functionality to the psilocybin bioproduction platform upon scale-up. Figure 14 shows psilocybin titer for each mutant as a function of time, with dashed lines representing the data recorded for the psilocybin production strain from Figure 4. Growth was consistent for each reactor as measured by ODeoo (data not shown), and differences in growth behavior were not found to be statistically significant (p>0.05).
[0147] It was observed that mutants KD10 and SH2 performed similarly to the psilocybin production strain until the end of the fermentation. Production was closely linked to growth,
SUBSTITUTE SHEET (RULE 26) with minimal increase in titers observed after stationary phase (t=40h) was reached (<100 mg/L). In contrast, the psilocybin production strain with methionine supplementation demonstrated continued psilocybin production even after the stationary phase was reached (>200 mg/L). The reasons for this behavior are unknown. Without wishing to be bound by theory, adding the accessory gene operon to the pETM6 plasmid could increase stress of maintaining the plasmid, encouraging plasmid loss.
[0148] Strain LD2 (with methionine supplementation) performed similarly to the psilocybin production strain in the non-supplemented condition. This may indicate that this particular mutant introduced a SAM-deficiency, rather than the intended purpose of increasing SAM pools. The poor performance of this mutant may not be due to the presence of luxS and mtn, but the particular pathway balancing between the accessory and psilocybin operons. To investigate this hypothesis, other mutants containing the luxS-mtn accessory operon should be scaled in bioreactor conditions.
Example 5: Strain Construction and Validation
Proposed Strain and Plasmid Libraries
[0149] A chart giving expected relationships between different upregulations and knockouts is shown in Table 5. Since mtn and luxS are demonstrated to improve vanillate titers when expressed together, these two genes will be paired when expressed on any plasmid. Similarly, cysE* and metA* will be expressed together. It can also be noted that since sahH offers an alternative pathway to luxS/mtn, it may be redundant to express these 3 genes together.
Table 5. Asterisk (*) represents genes which have enabled higher titers when expressed together in the literature. Cells labeled ‘A’ represent combinatorial expression that is hypothesized to be generally beneficial. Cells labeled ‘B’ represent combinatorial expression that is hypothesized to be redundant.
Figure imgf000037_0001
Table 6 represents a proposed plasmid list for intended overexpressions used in this project. Each of these plasmids is tested in a fermentation run with pETM6-SDM2x-PsiD-PsiK-PsiM, on the wild-type genomic background, LmetJ background, LspeD background, and
SUBSTITUTE SHEET (RULE 26) ΔmetJ ΔspeD background.
Plasmid pACM4-SDM2x-acs pACM4-SDM2x-sahH pACM4-SDM2x-luxS-mtn p ACM4 - SDM2 x-cy sE * -met A* pACM4-SDM2x-luxS-mtn-acs pACM4-SDM2x- cysE*-metA*-acs pACM4-SDM2x- cysE*-metA*-sahH pACM4-SDM2x- cysE*-metA*-acs-sahH
Strain Optimization
[0150] Once strains have been identified which result in either improved AMC dynamics or increased psilocybin production, these strains are run in scale-up studies using bench-scale bioreactors to test strains for decreased dependency on methionine. If the bioreactor experiments result in a reduced dependency on methionine supplementation, it can be assumed that AMC efficiency has been increased, and a promoter library for the psilocybin pathway will be tested on this genetic background to optimize psilocybin titers as described by Adams et al. Additionally, promoter libraries can be constructed for the overexpression plasmids used in that strain to further optimize metabolic efficiency and psilocybin production.
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12. Kunjapur, A. M., Hyun, J. C. & Prather, K. L. J. Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway. Microbial Cell Factories (2016) doi: 10. 1186/sl2934-016-0459-x.
13. Carhart-Harris, R. et al. Trial of Psilocybin versus Escitalopram for Depression. New England Journal of Medicine 384, (2021).
14. Daniel, J. & Haberman, M. Clinical potential of psilocybin as a treatment for mental health conditions. Mental Health Clinician 7, (2017).
15. Adams, A. M. et al. In vivo production of psilocybin in E. coli. Metabolic Engineering 56, (2019).
16. Gillam, E. M. J., Baba, T., Kim, B. R., Ohmori, S. & Guengerich, F. P. Expression of Modified Human Cytochrome P450 3A4 in Escherichia coli and Purification and Reconstitution of the Enzyme. Archives of Biochemistry and Biophysics 305, (1993).
17. Roe, A. J., O’Byme, C., McLaggan, D. & Booth, I. R. Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity. Microbiology 148, (2002).
18. Jones, J. A. et al. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Scientific Reports 5, (2015).
19. Jones, J. A. et al. Experimental and computational optimization of an Escherichia coli coculture for the efficient production of flavonoids. Metabolic Engineering 35, (2016).
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21. Bao, S.-H. et al. A dynamic and multilocus metabolic regulation strategy using quorum- sensing-controlled bacterial small RNA. Cell Reports 36, (2021).
22. Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. Angewandte Chemie International Edition 56, (2017).
23. Tuite, N. L., Fraser, K. R. & O’Byme, C. P. Homocysteine Toxicity in Escherichia coli Is Caused by a Perturbation of Branched-Chain Amino Acid Biosynthesis. Journal of Bacteriology 187, (2005).
24. Wolfe, A. J. The Acetate Switch. Microbiology and Molecular Biology Reviews 69, (2005).
25. Lin, H., Castro, N. M., Bennett, G. N. & San, K.-Y. Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering. Applied Microbiology and Biotechnology !]., (2006).
26. Farmer, W. R. & Liao, J. C. Reduction of aerobic acetate production by Escherichia coli. Applied and Environmental Microbiology 63, (1997).
27. Aristidou, A. A., San, K.-Y. & Bennett, G. N. Modification of central metabolic pathway inescherichia coli to reduce acetate accumulation by heterologous expression of thebacillus subtilis acetolactate synthase gene. Biotechnology and Bioengineering 44, (1994).
28. Halliday, N. M., Hardie, K. R., Williams, P., Winzer, K. & Barrett, D. A. Quantitative liquid chromatography-tandem mass spectrometry profiling of activated methyl cycle metabolites involved in LuxS-dependent quorum sensing in Escherichia coli. Analytical Biochemistry 403, (2010).
29. He, W. et al. Production of chondroitin in metabolically engineered E. coli. Metabolic Engineering 27, (2015).
Table 7: Sequences
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[0151] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.
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Claims

CLAIMS What is claimed is:
1. A method for the production of psilocybin or an intermediate or a side product thereof comprising: contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell, wherein the host cell is optionally cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, betaine and combinations thereof.
2. The method of claim 1, wherein the expression vector further comprises a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
3. The method of claim 1, further comprising contacting the prokaryotic cell with an expression vector comprising a gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
4. The method of claim 2, wherein the cysE is a feedback inhibition-resistant variant.
5. The method of claim 2, wherein the metA is a feedback inhibition-resistant variant.
6. The method of claim 1, wherein the prokaryotic host cell comprises a genomic knockout of metJ.
7. The method of claim 1, wherein the prokaryotic host cell comprises a genomic knockout of speD.
8. The method of claim 1, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
9. The method of claim 1, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
10. The method of claim 1, wherein the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
11. The method of claim 1, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
12. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
13. The method of claim 12, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
14. The method of claim 1, wherein the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
15. The method of claim 14, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
16. The method of any one of claims 12-15, wherein the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
17. The method of claim 16, wherein all accessory genes are under control of a single promoter in operon configuration, optionally wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
18. The method of claim 1, wherein the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamonium (4-OH-TMT).
19. The method of any one of claims 1-11, wherein the supplement does not comprise methionine.
20. The method of any one of claims 1-19, wherein the supplement is fed continuously to the host cell.
21. The method of any one of claims 1-20, wherein the host cell is grown in an actively growing culture.
22. A recombinant prokaryotic cell comprising: one or more expression vectors, wherein each expression vector comprises a gene selected from the group consisting of psiD, psiK, psiM, acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
23. The method of claim 22, wherein the cysE is a feedback inhibition-resistant variant.
24. The method of claim 22 or 23, wherein the metA is a feedback inhibition-resistant variant.
25. The method of any one of claims 22-24, wherein the prokaryotic host cell comprises a genomic knockout of metJ.
26. The method of any one of claims 22-25, wherein the prokaryotic host cell comprises a genomic knockout of speD.
27. The recombinant prokaryotic cell of claim 22, wherein the psiD gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
28. The recombinant prokaryotic cell of claim 22, wherein the psiK gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
29. The recombinant prokaryotic cell of claim 22, wherein the psiM gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
30. The recombinant prokaryotic cell of claim 22, wherein the acs gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
31. The recombinant prokaryotic cell of claim 22, wherein the luxS gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
32. The recombinant prokaryotic cell of claim 22, wherein the mtn gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
33. The recombinant prokaryotic cell of claim 22, wherein the SAM2 gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 37 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
34. The recombinant prokaryotic cell of claim 22, wherein the BHMT gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 39 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
35. The recombinant prokaryotic cell of claim 22, wherein the sahH gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
36. The recombinant prokaryotic cell of claim 22, wherein the cysE gene is cysE* and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
37. The recombinant prokaryotic cell of claim 22, wherein the metA gene is metA* and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
38. The recombinant prokaryotic cell of claim 22, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
39. The recombinant prokaryotic cell of claim 22, wherein the expression vector comprises a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
40. The recombinant prokaryotic cell of claim 39, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
41. The recombinant prokaryotic cell of claim 22, wherein the expression vector comprises a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon or monocistronic configuration.
42. The recombinant prokaryotic cell of claim 41, wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7,
Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
43. The recombinant prokaryotic cell of claim 22, wherein the expression vector further comprises an accessory gene selected from the group consisting of acs, luxS, mtn, sahH, cysE, metA, BHMT, SAM2, and combinations thereof.
44. The recombinant prokaryotic cell of claim 43, wherein all accessory genes are under control of a single promoter in operon configuration, optionally wherein the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
45. The recombinant prokaryotic cell claim 22, wherein the prokaryotic cell comprises a genomic knockout of metJ.
46. The recombinant prokaryotic cell of claim 22, wherein the prokaryotic cell comprises a genomic knockout of speD.
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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2008025744A1 (en) * 2006-08-28 2008-03-06 Novo Nordisk A/S Extra-cellular production of recombinant proteins
WO2013159055A1 (en) * 2012-04-20 2013-10-24 Codexis, Inc. Production of fatty alcohols from engineered microorganisms
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WO2021086513A1 (en) * 2019-10-28 2021-05-06 Miami University Methods for the production of psilocybin and intermediates or side products

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008025744A1 (en) * 2006-08-28 2008-03-06 Novo Nordisk A/S Extra-cellular production of recombinant proteins
WO2013159055A1 (en) * 2012-04-20 2013-10-24 Codexis, Inc. Production of fatty alcohols from engineered microorganisms
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