# US 11,441,164 B2 · Biosynthetic production of psilocybin and related intermediates in recombinant organisms

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- Number: US 11,441,164 B2
- Document ID: US11441164B2
- Status: Granted
- Kind: B2
- Application number: 17/099,539
- Filed: 2020-11-16
- Published: 2021-05-20
- Granted: 2022-09-13
- Earliest claimed: 2019-11-15
- Assignees: CB Therapeutics, Inc.
- Applicants: CB Therapeutics, INC.
- Inventors: Jacob Michael Vogan; Laura Flatauer Peiffer; James Lee Wade; Tyrone Jacob Yacoub; Kirsten Tang; Rachel Nadine Burnett
- Web page: https://patentgrep.com/patent/US11441164B2

## CPC

- C12N 15/81: for yeasts
- C12N 1/16: Yeasts; Culture media therefor
- C12N 9/0083: {Miscellaneous (1.14.99)}
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 9/1085: {transferring alkyl or aryl groups other than methyl groups (2.5)}
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 9/88: Lyases (4.)
- C12P 17/10: Nitrogen as only ring hetero atom
- C12Y 205/01054: 3-Deoxy-7-phosphoheptulonate synthase (2.5.1.54)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

The systems and methods herein include engineering a host to produce psilocybin using engineered enzymes, genetic changes, and exogenous psilocybin precursor addition (e.g., addition of L-tryptophan to a growing culture of a psilocybin producing recombinant host strain). The process occurs in genetically engineered host cell(s) that can produce psilocybin.

## Claims

### Claim 1

A recombinant host organism comprising:
a plurality of cells transfected by a gene expressed in the recombinant host organism;
wherein the recombinant host organism is a fungal species selected from the group consisting of Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica;
wherein the gene is codon optimized for expression in the recombinant host organism and is selected from a group consisting of PsiD, PsiH, PsiK, and PsiM; wherein:
PsiD encodes an L-tryptophan decarboxylase and comprises nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;
PsiH encodes a tryptamine 4-monooxygenase and comprises nucleic acid sequence SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6;
PsiK encodes a 4-hydroxytryptamine kinase and comprises nucleic acid sequence SEQ ID NO:7 or SEQ ID NO:8; and
PsiM encodes a methyl transferase and comprises codon optimized nucleic acid sequences SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

### Claim 2 (depends on 1)

The recombinant host organism of claim 1, comprising PsiD, PsiH, PsiK and PsiM, all codon optimized for expression in the recombinant host organism, wherein the organism synthesizes psilocybin.

### Claim 3 (depends on 2)

The recombinant host organism of claim 2, wherein the organism synthesizes psilocybin via at least a first pathway and a second pathway in sequential order; wherein:
the first pathway is a shikimate-chorismate pathway; and
the second pathway is an L-tryptophan pathway.

### Claim 4 (depends on 3)

The recombinant host organism of claim 3, wherein the synthesis of psilocybin via the first pathway and the second pathway increases titers of psilocybin in the plurality of cells over titers of psilocybin not synthesized via the first pathway and the second pathway.

### Claim 5 (depends on 3)

The recombinant host organism of claim 3, wherein the shikimate-chorismate pathway is modified by overexpression of at least one of an ARO1 gene, an ARO2 gene, an ARO3 gene, an ARO4 gene, or an AROL gene, and the L-tryptophan pathway is modified by overexpression of at least one of a TRP1 gene, a TRP2 gene, a TRP3 gene, or a TRP4 gene.

### Claim 6 (depends on 5)

The recombinant host organism of claim 5, wherein the first pathway is modified by expression of at least one of SEQ ID NO:27, SEQ ID NO 28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31 and the second pathway is modified by expression of at least one of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35.

### Claim 7 (depends on 3)

The recombinant host organism of claim 3, synthesizing at least one psilocybin intermediates selected from the group consisting of tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, and psilocin.

### Claim 8 (depends on 7)

The recombinant host organism of claim 7, wherein the synthesis of psilocybin via the first pathway and the second pathway increases a titer of a psilocybin intermediate in the plurality of cells over titers of the psilocybin intermediate not synthesized via the first pathway and the second pathway.

### Claim 9 (depends on 1)

The recombinant host organism of claim 1, further comprising a recombinant transporter protein that is codon optimized for expression in the recombinant host organism.

### Claim 10 (depends on 9)

The recombinant host organism of claim 9, wherein the recombinant transporter protein comprises SEQ ID NO:36.

### Claim 11 (depends on 1)

The recombinant host organism of claim 1, growing in a medium comprising glucose, galactose, sucrose, fructose, molasses, or any combination thereof.

### Claim 12 (depends on 2)

A method, the method comprising:
transfecting a plurality of cells in a recombinant host organism a set of genes comprising PsiD, PsiH, PsiK and PsiM, creating the recombinant host organism of claim 2; and
synthesizing psilocybin in the recombinant host organism.