Aromatic prenyltransferase from cannabis

Patent metadata

Field	Value
Publication number	US-9765308-B2
Application number	US-201414510232-A
Country	US
Kind code	B2
Filing date	Oct 9, 2014
Priority date	Aug 12, 2009
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

Nucleic acid molecules from cannabis has been isolated and characterized and encode polypeptides having aromatic prenyltransferase 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.

First claim

The invention claimed is:

1. An isolated or purified nucleic acid molecule comprising a nucleotide sequence having:
i) at least 95% sequence identity to SEQ ID NO: 1,
ii) the sequence as set forth in SEQ ID NO:1,
iii) a nucleotide sequence encoding an isolated polypeptide having at least 95% sequence identity to SEQ ID NO: 2,
iv) a nucleotide sequence encoding an isolated polypeptide having the sequence as set out in SEQ ID NO: 2 or a conservatively substituted sequence thereof; or
v) a codon degenerate sequence of i), ii), iii) or iv),

wherein i), ii), iii), iv) and v) encode a polypeptide having aromatic prenyltransferase activity and wherein the nucleic acid molecule is a cDNA and/or contains at least one non-naturally occurring substitution modification relative to SEQ ID NO:1.

2. An isolated or purified polypeptide comprising an amino acid sequence having:
i) at least 95% sequence identity to SEQ ID NO: 2,
ii) the sequence as set out in SEQ ID NO: 2, or
iii) a conservatively substituted amino acid sequence of i) or ii);

having aromatic prenyltransferase activity and containing at least one non-naturally occurring substitution modification relative to SEQ ID NO:2.

3. A vector, construct or recombinant expression system comprising a nucleic acid molecule comprising:
i) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 1,
ii) a nucleotide sequence having the sequence as set forth in SEQ ID NO:1,
iii) a nucleotide sequence encoding an isolated polypeptide having at least 95% sequence identity to SEQ ID NO: 2,
iv) a nucleotide sequence encoding an isolated polypeptide having the sequence as set out in SEQ ID NO: 2; or
v) a codon degenerate sequence of any one of i) to iv),

wherein i), ii), iii), iv) and v) encode a polypeptide having aromatic prenyltransferase activity.

4. An in vitro process of transferring a prenyl group comprising: reacting a prenyl group acceptor molecule with a prenyl group donor molecule in the presence of an aromatic prenyltransferase polypeptide comprising an amino acid sequence having:
i) at least 95% sequence identity to SEQ ID NO: 2,
ii) the sequence as set out in SEQ ID NO: 2, or
iii) a conservatively substituted amino acid sequence of i) or ii);

wherein the aromatic prenyltransferase polypeptide catalyzes the transfer of the prenyl group from the prenyl group donor molecule to the prenyl group acceptor molecule.

5. A method of decreasing levels of cannabinoid compounds in a cannabis plant, cannabis cell or cannabis tissue comprising expressing the nucleic acid molecule as defined in claim 1 i) to iii) or a part thereof to silence expression of the aromatic prenyltransferase gene in the cannabis plant, cannabis cell or cannabis tissue, in comparison to a cannabis plant, cannabis cell or cannabis tissue grown without the use of the nucleic acid molecule for silencing.

6. An in vitro method of increasing levels of cannabinoid compounds in a cell comprising expressing or over-expressing the nucleic acid molecule as defined in claim 1 i) to v) in the cell, in comparison to a cell of the same species not expressing or over-expressing the nucleic acid molecule.

7. An in vitro method of increasing levels of cannabinoid compounds in a cell comprising expressing or over-expressing a nucleic acid molecule encoding the polypeptide as defined in claim 2 in the cell, in comparison to a cell of the same species not expressing or over-expressing the nucleic acid molecule.

8. The method of claim 6 , wherein the cell is or is from a microorganism, an insect cell or a plant cell.

9. The method of claim 8 , wherein the microorganism is yeast or E. coli or the insect cell is a Spodoptera frugiperda cell.

10. The method of claim 6 , 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.

11. The method of claim 10 , wherein the one or more enzymes in the cannabinoid biosynthetic pathway is one or more of Δ 9 -tetrahydrocannabinolic acid synthase, cannabidiolic acid synthase and cannabichromenic acid synthase.

12. The process of claim 4 , wherein the aromatic prenyltransferase is in a membrane preparation.

13. The process of claim 12 , wherein the membrane preparation is obtained by expressing the aromatic prenyltransferase in a cell and obtaining the membrane fraction of the cell, optionally by sonicating the cell.

14. The process of claim 13 , wherein the cell is a yeast cell or an insect cell.

15. The process of claim 13 , wherein the step of expressing the aromatic prenyltransferase comprises transforming the cell with a vector, construct or virus.

16. The process of claim 7 , wherein the prenyl group donor molecule is geranyl disphosphate and/or the prenyl group acceptor molecule is olivetolic acid.

17. The process of claim 12 , wherein the membrane preparation further comprises one or more other enzymes in a cannabinoid biosynthetic pathway.

18. The process of claim 17 , wherein the one or more other enzymes in a cannabinoid biosynthetic pathway is one or more of Δ 9 -tetrahydrocannabinolic acid synthase, cannabidiolic acid synthase and cannabichromenic acid synthase.

19. A membrane preparation comprising the polypeptide of claim 2 .

20. The membrane preparation of claim 19 , prepared from a recombinant cell expressing the polypeptide.

21. A cell expressing the polypeptide of claim 2 .

22. A recombinant cell comprising the vector, construct or recombinant expression system of claim 3 .

Assignees

• Nat Res Council Canada
• Univ Saskatchewan
• The Univ Of Saskatchewan

Inventors

• Page Jonathan E
• Boubakir Zakia

Classifications

• C12P17/06

  containing a six-membered hetero ring, e.g. fluorescein · CPC title

• C12Y205/01

  transferring alkyl or aryl groups, other than methyl groups (2.5.1) · CPC title

• C12N15/8243

  involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine · CPC title

• C12P7/22

  aromatic · CPC title

• C12N9/1085 Primary

  transferring alkyl or aryl groups other than methyl groups (2.5) · CPC title