Biosynthetic production of psilocybin and related intermediates in recombinant organisms

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.

1 - 21 . (canceled) 
     
     
         22 . A recombinant host organism comprising:
 a plurality of cells transfected by a non-natural 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 non-natural gene is selected from a group consisting of PsiD, PsiH, PsiK, and PsiM, wherein:   PsiD encodes an L-tryptophan decarboxylase comprising an amino acid sequence having at least 90% identity to any one of amino acid sequences set forth in SEQ ID NO:14-SEQ ID NO:16;   PsiH encodes a tryptamine 4-monooxygenase comprising an amino acid sequence having at least 90% identity to any one of amino acid sequences set forth in SEQ ID NO:17-SEQ ID NO:19;   PsiK encodes a 4-hydroxytryptamine kinase comprising an amino acid sequence having at least 90% identity to any one of amino acid sequences set forth in SEQ ID NO:20 or SEQ ID NO:21; and   PsiM encodes a methyl transferase comprising an amino acid sequence having at least 90% identity to any one of amino acid sequences set forth in SEQ ID NO:22-SEQ ID NO:26.   
     
     
         23 . The recombinant host organism of  claim 22 , wherein
 the L-tryptophan decarboxylase comprises any one of amino acid sequences set forth in SEQ ID NO:14-SEQ ID NO:16;   the tryptamine 4-monooxygenase comprises any one of amino acid sequences set forth in SEQ ID NO:17-SEQ ID NO:19;   the 4-hydroxytryptamine kinase comprises any one of amino acid sequences set forth in SEQ ID NO:20 or SEQ ID NO:21; and   the methyl transferase comprises any one of amino acid sequences set forth in SEQ ID NO:22-SEQ ID NO:26.   
     
     
         24 . The recombinant host organism of  claim 22 , comprising PsiD, PsiH, PsiK and PsiM, wherein the organism synthesizes psilocybin. 
     
     
         25 . The recombinant host organism of  claim 22 , further comprising at least one non-natural gene selected from the group consisting of: SEQ ID NO:27-SEQ ID NO:35. 
     
     
         26 . The recombinant host organism of  claim 22 , further comprising a recombinant transporter protein that is codon optimized for expression in the recombinant host organism. 
     
     
         27 . The recombinant host organism of  claim 26 , wherein the recombinant transporter protein comprises SEQ ID NO:36. 
     
     
         28 . The recombinant host organism of  claim 22 , growing in a medium comprising glucose, galactose, sucrose, fructose, molasses, or any combination thereof. 
     
     
         29 . A method, the method comprising:
 transfecting a plurality of cells in a recombinant host organism with a set of genes comprising PsiD, PsiH, PsiK and PsiM, creating the recombinant host organism of  claim 22 ; and   synthesizing psilocybin in the recombinant host organism.