CANNABIDIOLIC ACID (CBDA) SYNTHASE VARIANTS, AND MANUFACTURE AND USE THEREOF
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 of SEQ ID NOs: 2-45 or a polynucleotide sequence with at least 90%, 95%, 98%, 99% or 99.9% identity with any one of SEQ ID NOs: 2-45. 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.
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: 72-115.
2 . The recombinant vector of claim 1 , wherein the polynucleotide sequence encodes a polypeptide as set forth in any one of SEQ ID NOs: 72-115.
3 . The recombinant vector of claim 1 , wherein the polynucleotide sequence comprises the sequence as set forth in any one of SEQ ID NOs: 2-45, 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: 2-45.
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: 59-68.
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: 59-68.
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: 46-55 and 130.
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: 46-55 and 130.
11 . The recombinant vector of claim 1 , comprising a purification/detection tag as set forth in any one of SEQ ID NOs: 56-58 and 129.
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 R 2 is a linear or branched CH 3 , C 2 H 5 , C 3 H 7 , C 4 H 9 , C 5 H 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: 72-115 or an amino acid sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs: 72-115.
18 . The polypeptide of claim 17 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 83, 86, 92, or 97 or an amino acid sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to SEQ ID NO: 83, 86, 92, or 97.
19 . A nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 72-115 or an amino acid sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs: 72-115.
20 . The nucleic acid of claim 19 , wherein the nucleic acid comprises the polynucleotide sequence of any one of SEQ ID NOs: 2-45 or a polynucleotide sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to any one of SEQ ID NOs: 2-45.
21 . The nucleic acid of claim 20 , wherein the nucleic acid comprises the polynucleotide sequence of SEQ ID NO: 13, 16, 22, or 27 or a polynucleotide sequence comprising at least 90%, 95%, 98%, or 99.9% sequence identity to SEQ ID NO: 13, 16, 22, or 27.