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 . A host cell that comprises a heterologous polynucleotide encoding a polyketide synthase (PKS), wherein the PKS comprises an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 7, 15, 145, and 714, or wherein the PKS comprises a conservatively substituted version of any one of SEQ ID NOs: 7, 15, 145, and 714, and wherein the host cell further comprises one or more heterologous polynucleotides encoding one or more of: a polyketide cyclase (PKC), a prenyltransferase (PT), and/or a terminal synthase (TS). 
     
     
         2 . The host cell of  claim 1 , 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. 
     
     
         3 . The host cell of  claim 1 , 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 SEO 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.   
     
     
         4 - 71 . (canceled) 
     
     
         72 . The host cell of  claim 1 , wherein the host 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; and
 wherein R is straight-chain unsubstituted C 1-20  alkyl. 
 
     
     
         73 . The host cell of  claim 72 , wherein the host cell is capable of producing 3,5,7-trioxododecanoyl-CoA, olivetol, olivetolic acid, cannabigerolic acid (8a), cannabidiolic acid (9a), tetrahydrocannabinolic acid (10a), and/or cannabichromenic acid (11a). 
     
     
         74 . The host cell of  claim 73 , wherein the host cell produces more 3,5,7-trioxododecanoyl-CoA, olivetol, and/or olivetolic acid than a host cell that: (i) does not comprise the PKS that comprises the amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 7, 15, 145, and 714 and (ii) comprises a heterologous polynucleotide encoding a PKS that comprises the amino acid sequence of SEQ ID NO: 5. 
     
     
         75 . The host cell of  claim 1 , wherein the PKS comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 7: V71Y and F70M. 
     
     
         76 . The host cell of  claim 1 , 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 host cell of  claim 1 , wherein the PKS comprises the amino acid sequence of any one of SEQ ID NOs: 7, 15, 145, and 714. 
     
     
         78 . The host cell of  claim 1 , wherein the heterologous polynucleotide comprises a nucleotide sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 38, 175, 176, and 205, or a codon degenerate nucleotide sequence thereof. 
     
     
         79 . The host cell of  claim 1 , wherein the host cell is a yeast cell. 
     
     
         80 . The host cell of  claim 79 , wherein the yeast cell is a  Saccharomyces  cell, a  Yarrowia  cell, a  Pichia  cell or a  Komagataella  cell. 
     
     
         81 . The host cell of  claim 80 , wherein the  Saccharomyces  cell is a  Saccharomyces cerevisiae  cell. 
     
     
         82 . The host cell of  claim 1 , wherein the PKC is an olivetolic acid cyclase (OAC). 
     
     
         83 . The host cell of  claim 72 , wherein R is a straight-chain unsubstituted C 1-10  alkyl. 
     
     
         84 . The host cell of  claim 72 , wherein R is a straight-chain unsubstituted C 3  or C 5  alkyl. 
     
     
         85 . A method comprising culturing the host cell of  claim 1 . 
     
     
         86 . A method for producing a cannabinoid compound or a cannabinoid precursor, wherein the method comprises culturing a host cell that comprises a heterologous polynucleotide encoding a polyketide synthase (PKS), wherein the PKS comprises an amino acid sequence that has at least 90% identity to SEQ ID NO: 7, 15, 145, or 714 and 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; and
 wherein R is straight-chain unsubstituted C 1-20  alkyl. 
 
     
     
         87 . A bioreactor for producing a cannabinoid compound or a cannabinoid precursor containing:
 a. malonyl-CoA;   b. an optionally substituted alkanoic acid; and   c. a polyketide synthase (PKS) comprising an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 7, 715, 145, and 714, or a conservatively substituted version thereof.   
     
     
         88 . The bioreactor of  claim 87 , further comprising an acyl activating enzyme (AAE), a polyketide cyclase (PKC), a prenyltransferase (PT), and/or a terminal synthase (TS).