Cannabichromenic acid synthase from cannabis sativa
Nucleic acid molecules from cannabis have been isolated and characterized and encode polypeptides having cannabichromenic acid synthase activity. Expression or over-expression of the nucleic acids alters levels of cannabinoid compounds. The polypeptides may be used in vivo or in vitro to produce cannabinoid compounds.
1. A method of modulating levels of cannabinoid compounds in an organism, cell or tissue, said method comprising:
a) introducing a nucleic acid molecule comprising:
i) a nucleotide sequence complementary to a polynucleotide encoding at least a portion of a polypeptide having the sequence set for in SEQ ID NO: 2 or 6 or a polypeptide having at least 99.5% sequence identity to SEQ ID NO: 2 or 6; or
ii) a nucleotide sequence complementary to a polynucleotide having at least, greater than or about 99.5% sequence identity to at least a portion of SEQ ID NO: 1 or 5;
into the organism, cell or tissue to decrease the level of a cannabinoid compound,
wherein the portion comprises greater than, at least or about 15 contiguous nucleotides,
and wherein the nucleic acid molecule is comprised in a construct, to silence cannabichromenic acid synthase gene in the organism, cell or tissue to decrease the level of a cannabinoid compound; or
b) introducing a nucleic acid molecule or construct comprising said nucleic acid molecule, and expressing or over-expressing a polypeptide having cannabichromenic acid synthase activity encoded by said nucleic acid molecule, said nucleic acid molecule comprising
i) a nucleotide sequence encoding a polypeptide having the sequence set for in SEQ ID NO: 2 or 6 or a polypeptide having at least 99.5% sequence identity to SEQ ID NO: 2 or 6;
ii) a nucleotide sequence having at least, greater than or about 99.5% sequence identity to SEQ ID NO: 1 or 5; or
iii) a nucleotide sequence having at least, greater than or about 90% sequence identity to SEQ ID NO: 8 or 9;
in the organism, cell or tissue to increase the level of a cannabinoid compound.
2. The method of claim 1 , wherein the organism is a microorganism.
3. The method of claim 2 , wherein the microorganism is yeast or E. coli.
4. The method of claim 1 b) wherein the nucleic acid molecule is expressed or over-expressed in combination with expression or over-expression of one or more other nucleic acids that encode one or more enzymes in a cannabinoid biosynthetic pathway.
5. The method of claim 4 , wherein the one or more enzymes in a cannabinoid biosynthetic pathway is one or more of hexanoyl CoA synthetase, olivetolic acid cyclase, a THCA synthase, a CBDA synthase, or aromatic prenyltransferase PT1.
6. The method of claim 1 , wherein one or more of the cannabinoids are modulated, optionally cannabigerolic acid, cannabigerol, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, Δ9-tetrahydrocannabinol, cannabidiol, cannabichromene and cannabidiol.
7. The method of claim 1 b), wherein the nucleotide sequence is as set forth in SEQ ID NO: 1, or 5 or a codon degenerate nucleotide sequence thereof, optionally wherein the codon degenerate nucleotide sequence comprises the sequence set forth in SEQ ID NO: 8 or 9, or the complement of any of the foregoing having cannabichromenic acid synthase activity.
8. The method of claim 1 , the nucleic acid molecule is coupled to one or more heterologous moieties, optionally one or more of a linker, a signal sequence, a detectable label or a tag.