Biosynthesis of cannabinoids and cannabinoid precursors

Abstract

Aspects of the disclosure relate to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro.

1 .- 71 . (canceled) 
     
     
         72 . A yeast cell for producing a cannabinoid compound or a cannabinoid precursor that comprises a heterologous polynucleotide encoding a polyketide synthase (PKS), wherein the PKS comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 7 or wherein the PKS comprises a conservatively substituted version of SEQ ID NO: 7. 
     
     
         73 . The yeast cell of  claim 72 , wherein relative to the sequence of SEQ ID NO: 7, the PKS comprises an amino acid substitution at a residue corresponding to position 28, 34, 50, 70, 71, 76, 88, 100, 151, 203, 219, 285, 359, and/or 385 in SEQ ID NO: 7. 
     
     
         74 . The yeast cell of  claim 72 , wherein the PKS comprises:
 a) the amino acid P at a residue corresponding to position 28 in SEQ ID NO: 7;   b) the amino acid Q at a residue corresponding to position 34 in SEQ ID NO: 7;   c) the amino acid N at a residue corresponding to position 50 in SEQ ID NO: 7;   d) the amino acid M at a residue corresponding to position 70 in SEQ ID NO: 7;   e) the amino acid Y at a residue corresponding to position 71 in SEQ ID NO: 7;   f) the amino acid I at a residue corresponding to position 76 in SEQ ID NO: 7;   g) the amino acid A at a residue corresponding to position 88 in SEQ ID NO: 7;   h) the amino acid P or T at a residue corresponding to position 100 in SEQ ID NO: 7;   i) the amino acid P at a residue corresponding to position 151 in SEQ ID NO: 7;   j) the amino acid K at a residue corresponding to position 203 in SEQ ID NO: 7;   k) the amino acid C at a residue corresponding to position 219 in SEQ ID NO: 7;   l) the amino acid A at a residue corresponding to position 285 in SEQ ID NO: 7;   m) the amino acid M at a residue corresponding to position 359 in SEQ ID NO: 7; and/or   n) the amino acid M at a residue corresponding to position 385 in SEQ ID NO: 7.   
     
     
         75 . The yeast cell of  claim 72 , wherein the PKS comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 7: V71Y and/or F70M. 
     
     
         76 . The yeast cell of  claim 72 , wherein the PKS comprises:
 a) C at a residue corresponding to position 164 in SEQ ID NO: 7;   b) H at a residue corresponding to position 304 in SEQ ID NO: 7; and/or   c) N at a residue corresponding to position 337 in SEQ ID NO: 7.   
     
     
         77 . The yeast cell of 72, wherein the heterologous polynucleotide comprises a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 38 or 176. 
     
     
         78 . The yeast cell of  claim 72 , wherein the yeast cell is a  Saccharomyces  cell, a  Yarrowia  cell, a  Pichia  cell or a  Komagataella  cell. 
     
     
         79 . The yeast cell of  claim 78 , wherein the yeast cell is a  Saccharomyces  cell. 
     
     
         80 . The yeast cell of  claim 79 , wherein the  Saccharomyces  cell is a  Saccharomyces cerevisiae  cell. 
     
     
         81 . The yeast cell of  claim 78 , wherein the yeast cell is a  Yarrowia  cell. 
     
     
         82 . The yeast cell of  claim 72 , wherein the yeast cell further comprises one or more heterologous polynucleotides encoding one or more of: an acyl activating enzyme (AAE), a polyketide cyclase (PKC), a prenyltransferase (PT), and/or a terminal synthase (TS). 
     
     
         83 . The yeast cell of  claim 72 , wherein the yeast cell is capable of producing a cannabinoid compound or a cannabinoid precursor, wherein the cannabinoid compound is a compound of Formulas 8, 9, 10, or 11: 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof;
 wherein the cannabinoid precursor is a compound of Formulas 4, 5, or 6: 
 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof;
 wherein R is straight-chain unsubstituted C 1-20  alkyl, 
 
     
     
         84 . The yeast cell of  claim 72 , wherein the yeast cell is capable of producing 3,5,7-trioxododecanoyl-CoA, olivetol, olivetolic acid, cannabigerolic acid (8α), cannabidiolic acid (9a), tetrahydrocannabinolic acid (10α), and/or cannabichromenic acid (11a). 
     
     
         85 . The yeast cell of  claim 84 , wherein the yeast cell is capable of producing more 3,5,7-trioxododecanoyl-CoA, olivetol, and/or olivetolic acid than a yeast cell that expresses a PKS that comprises SEQ ID NO: 5. 
     
     
         86 . The yeast cell of  claim 82 , wherein the PKC is an olivetolic acid cyclase (OAC). 
     
     
         87 . The yeast cell of  claim 82 , wherein the PT is a cannabigerolic acid synthase (CBGAS). 
     
     
         88 . The yeast cell of  claim 82 , wherein the TS is a tetrahydrocannabinolic acid synthase (THCAS), a cannabidiolic acid synthase (CBDAS), and/or a cannabichromenic acid synthase (CBCAS). 
     
     
         89 . The yeast cell of  claim 72 , wherein the PKS comprises SEQ ID NO: 7.