Tetrahydrocannabinolic acid (thca) synthase variants, and manufacture and use thereof

Abstract

Provided herein is a recombinant vector that comprises a promoter, a secretion tag gene of interest (GOI), and optionally a purification and/or a detection tag. The GOI comprises a polynucleotide sequence as set forth in any one of SEQ ID NOs: 2-57 or a polynucleotide sequence with at least 90%, 95%, 98%, 99% or 99.9% identity with any one of SEQ ID NOs: 2-57. Also, disclosed are the sequences of polypeptides that result from expression of a GOI by a host expression system, and a method of producing cannabinoid compounds.

We claim: 
     
         1 . A recombinant vector comprising a promoter, secretion tag, gene of interest (GOI), and optionally a purification and/or a detection tag, wherein the gene of interest comprises a polynucleotide sequence encoding a polypeptide with at least 90%, 95%, 98%, or 99.9% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 84-139. 
     
     
         2 . The recombinant vector of  claim 1 , wherein the polynucleotide sequence encodes a polypeptide as set forth in any one of SEQ ID NOs: 84-139. 
     
     
         3 . The recombinant vector of  claim 1 , wherein the polynucleotide sequence comprises the sequence as set forth in any one of SEQ ID NOs: 2-57, or a sequence comprising at least 90%, 95%, 98%, 99%, or 99.9% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 2-57. 
     
     
         4 . The recombinant vector of  claim 1 , wherein the gene of interest is operably linked to a promoter. 
     
     
         5 . The recombinant vector of  claim 4 , wherein the promoter is a polynucleotide as set forth in any one of SEQ ID NOs: 71-80. 
     
     
         6 . The recombinant vector of  claim 5 , wherein the promoter has a polynucleotide sequence comprising at least 90%, 95%, 98%, or 99% sequence identity to the sequence of SEQ ID NOs: 71-80. 
     
     
         7 . The recombinant vector of  claim 4 , wherein the promoter is a constitutive promoter or an inducible promoter. 
     
     
         8 . The recombinant vector of  claim 4 , wherein the promoter is selected from a group consisting of an alcohol oxidase 1 promoter (pAOX1), an alcohol oxidase 2 promoter (pAOX2), a dihydroxyacetone kinase promoter (pDAK), an S-hydroxymethyl-glutathione hydrolase promoter (pFGH), an NAD + -dependent formylglutathione dehydrogenase promoter (pFDH), a fructose 1,6-bisphosphate aldolase promoter (pFBA2), a peroxisomal membrane signal receptor PTS1 promoter (pPEX5), a peroxisomal protein Pex8p promoter (pPEX8), an alcohol dehydrogenase 2 promoter (pADH2) and a peroxin Pex14p promoter (pPEX14). 
     
     
         9 . The recombinant vector of  claim 1 , comprising a secretion tag as set forth in any one of SEQ ID NOs: 58-67 and 154. 
     
     
         10 . The recombinant vector of  claim 9 , wherein the sequence of the secretion tag is at least 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs: 58-67 and 154. 
     
     
         11 . The recombinant vector of  claim 1 , comprising a purification tag as set forth in any one of SEQ ID NOs: 68-70 and 153. 
     
     
         12 . The recombinant vector of  claim 11 , wherein the purification/detection tag is selected from the group consisting of a human influenza hemagglutinin (HA) tag, a His 6 tag and an HN tag. 
     
     
         13 . A host expression system comprising the recombinant vector of  claim 1 . 
     
     
         14 . The host expression system of  claim 13 , comprising a microbial cell, a yeast cell, a plant cell, or an animal cell. 
     
     
         15 . The host expression system of  claim 14 , wherein the yeast cell is selected from the group consisting of  Pichia pastoris, Pichia angusta, Pichia guillermordii, Pichia methanolica, Pichia inositovera, Hansenula polymorpha, Candida boidinii , and  Yarrowia lipolytica.    
     
     
         16 . A method of producing a cannabinoid compound, comprising reacting a cannabinoid precursor with an enzyme that is expressed by the host expression system of  claim 13 , wherein the cannabinoid precursor is compound of Formula I (see below): wherein R 1  is H or —COOH and R2 is a linear or branched CH 3 , C 2 H 5 , C 3 H 7 , C 4 H 9 , C 5   10 , C 6 H 13 , C 7 H 15  or C 8 H 17  group. 
       
         
           
           
               
               
           
         
       
     
     
         17 . A polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 84-139 or an amino acid sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs: 84-139. 
     
     
         18 . The polypeptide of  claim 17 , wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 91 and 110-116 or an amino acid sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs: 91 and 110-116. 
     
     
         19 . A nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 84-139 or an amino acid sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs:
 84-139. The nucleic acid of  claim 19 , wherein the nucleic acid comprises the polynucleotide sequence of any one of SEQ ID NOs: 2-57 or a polynucleotide sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs: 2-57.   
     
     
         21 . The nucleic acid of claim  20 , wherein the nucleic acid comprises the polynucleotide sequence of any one of SEQ ID NOs: 9 and 28-34 or a polynucleotide sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs: 9 and 28-34.