Genes and proteins for aromatic polyketide synthesis

Patent metadata

Field	Value
Publication number	US-9611460-B2
Application number	US-201113641272-A
Country	US
Kind code	B2
Filing date	Apr 15, 2011
Priority date	Apr 15, 2010
Publication date	Apr 4, 2017
Grant date	Apr 4, 2017

Abstract

Nucleic acid molecules encoding polypeptides having polyketide synthase activity have been identified and characterized. Expression or over-expression of the nucleic acids alters levels of cannabinoid compounds in organisms. 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 cDNA nucleic acid molecule consisting of: i) a nucleotide sequence as set forth in SEQ ID NO: 1 or 3 or a codon degenerate nucleotide sequence thereof; ii) a nucleotide sequence of 303 nucleotides that encodes a polypeptide with an amino acid sequence of SEQ ID NO:2 or a conservatively substituted amino acid sequence thereof; or iii) a fragment of i) or ii) having a length of at least 300 base pairs; wherein the nucleic acid molecule encodes a polypeptide with polyketide cyclase activity.

2. The nucleic acid molecule of claim 1 , wherein the nucleotide sequence is as set forth in SEQ ID NO: 1 or a codon degenerate nucleotide sequence thereof.

3. The isolated or purified cDNA nucleic acid molecule of claim 1 , wherein the nucleotide sequence has i) the sequence of SEQ ID NO: 1 or 3; or ii) a fragment thereof having a length of at least 300 base pairs.

4. An isolated or purified polypeptide consisting of: i) an amino acid sequence having one amino acid difference from the sequence set forth in SEQ ID NO:2; ii) a conservatively substituted amino acid sequence of the sequence set out in SEQ ID NO:2; iii) any of i) or ii) tagged with a HIS tag; or iv) an amino acid sequence as set forth in SEQ ID NO:2 tagged with a HIS tag.

5. The polypeptide of claim 4 , wherein the amino acid sequence is as set forth in SEQ ID NO: 2 or is a conservatively substituted amino acid sequence thereof and the polypeptide is tagged with a HIS tag.

6. The polypeptide of claim 4 having polyketide cyclase activity.

7. A vector, construct or expression system comprising the nucleic acid molecule of claim 1 .

8. A host cell transformed with the nucleic acid molecule of claim 1 .

9. A process of altering levels of cannabinoid compounds in a cannabis plant, cannabis cell or cannabis tissue comprising introducing a nucleic acid molecule using a RNAi construct, a miRNA, VIGS virus, antisense oligonucleotide, a targeted mutagenesis construct or using Targeting Induced Local Lesions IN Genomes (TILLING) wherein the nucleic acid molecule or TILLING inhibits expression of SEQ ID NO:1 or a part thereof encoding a polypeptide with polyketide cyclase activity that catalyzes synthesis of an aromatic polyketide, wherein the altered levels of cannabinoid compounds is in comparison to cannabis plant, cannabis cell or cannabis tissue of the same species grown under similar conditions but that does not comprise the nucleic acid molecule for inhibiting SEQ ID NO: 1 or part thereof, or that has not been subjected to TILLING.

10. A process of altering levels of cannabinoid compounds in an organism, cell or tissue comprising introducing and expressing or over-expressing a nucleic acid molecule encoding a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 or a conservatively substituted amino acid sequence of the sequence set forth in SEQ ID NO: 2, and having polyketide cyclase activity in the organism, cell or tissue, wherein the expressing or over-expression is in comparison to an organism, cell or tissue of the same species grown under similar conditions but without the introducing and expressing or over-expressing of the nucleic acid molecule.

11. The process of claim 10 , wherein the organism, or cell, is a microorganism.

12. The process of claim 11 , wherein the microorganism is Saccharomyces cerevisiae yeast or E. coli.

13. The process of claim 10 , 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.

14. The process of claim 13 , wherein the one or more enzymes in a cannabinoid biosynthetic pathway is one or more of an acyl CoA synthetase, a type III polyketide synthase, a polyketide cyclase, an aromatic prenyltransferase or a cannabinoid-forming oxidocylase.

15. The process of claim 14 , wherein the one or more enzymes in a cannabinoid biosynthetic pathway is one or more of a hexanoyl CoA synthetase, a type III polyketide synthase/olivetol synthase, a geranylpyrophosphate:olivetolate geranyltransferase, a Δ 9 -tetrahydrocannabinolic acid synthase, a cannabidiolic acid synthase or a cannabichromenic acid synthase.

16. The process of claim 9 , wherein the cannabinoid compound is one or more of cannabigerolic acid, Δ 9 -tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, Δ 9 -tetrahydrocannabinol, cannabidiol or cannabichromene or an analog thereof comprising a side-chain of 1 to 9 carbon atoms in length.

17. The process of claim 10 , wherein the cannabinoid compound is one or more of cannabigerolic acid, Δ 9 -tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, Δ 9 -tetrahydrocannabinol, cannabidiol or cannabichromene or an analog thereof comprising a side-chain of 1 to 9 carbon atoms in length.

18. The polypeptide of claim 4 comprising a conservatively substituted amino acid sequence of the sequence set forth in SEQ ID NO:2, wherein the polypeptide is optionally tagged with a HIS tag.

19. The vector, construct or expression system of claim 7 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 1or 3 or a fragment thereof having at least 300 base pairs.

20. The host cell of claim 8 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 1 or 3 or a fragment thereof having at least 300 base pairs.

21. The host cell of claim 8 , wherein the cell is a yeast cell or a bacteria.

22. The host cell of claim 20 , wherein the cell is a yeast cell or a bacteria.

23. The process of claim 9 , wherein the process comprises introducing a nucleic acid molecule using the RNAi construct or the targeted mutagenesis construct.

24. The process of claim 10 , wherein the nucleic acid molecule has a sequence of SEQ ID NO: 1 or 3.

25. The process of claim 11 , wherein the microorganism is yeast or bacteria.

Assignees

• Page Jonathan E
• Gagne Steve
• Nat Res Council Canada
• Univ Saskatchewan

Inventors

• Page Jonathan E
• Gagne Steve

Classifications

• C12Y203/01206

  3,5,7-Trioxododecanoyl-CoA synthase (2.3.1.206) · CPC title

• C12P17/06 Primary

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

• C12P7/22

  aromatic · CPC title

• C12Y404/01026

  Olivetolic acid cyclase (4.4.1.26) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title