Fragrance and methods for production of 5-epi-β-vetivone, 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one, and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one
The present invention is directed to novel methods for production of 5-epi-β-vetivone, 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and 2-isopropyl-6,10-dimethylspiro[4.5]deca-1,6-dien-8-one, which are useful for their fragrant qualities. Provided are methods for production of premnaspirodiene from a terpene substrate, methods for production of 5-epi-β-vetivone from premnaspirodiene as starting material, and methods for production of 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one from premnaspirodiene as starting material.
1. A method for producing (−)-premnaspirodiene from a terpene substrate, comprising the steps of:
(a) providing a terpene substrate to a host cell transformed or transfected with a vector comprising a sequence of nucleic acid residues encoding a premnaspirodiene synthase gene;
(b) culturing the host cell by fermentation under conditions suitable to produce (−)-premnaspirodiene from the terpene substrate, wherein:
the fermentation system comprises an aqueous and a non-polar solvent phase;
the non-polar solvent phase is substantially immiscible with the aqueous phase; and
the premnaspirodiene is sequestered in the non-polar solvent phase during fermentation; and
(c) isolating the (−)-premnaspirodiene from the non-polar solvent phase.
2. The method of claim 1 , wherein the step of providing the terpene substrate is performed by synthesis of the terpene substrate by the host cell.
3. The method of claim 2 , wherein the terpene substrate is farnesyl diphosphate.
4. The method of claim 1 , wherein the host cell is a eukaryotic cell.
5. The method of claim 4 , wherein the host cell is a yeast cell.
6. The method of claim 5 , wherein the host cell is a Saccharomyces cerevisiae cell.
7. A method for producing (−)-premnaspirodiene from a terpene substrate, comprising the steps of:
(a) providing a terpene substrate to a host cell transformed or transfected with a vector comprising a sequence encoding a premnaspirodiene synthase gene;
(b) culturing the host cell under conditions suitable to produce (−)-premnaspirodiene from the terpene substrate, wherein the (−)-premnaspirodiene is sequestered as it is produced during the culturing step; and
(c) isolating the (−)-premnaspirodiene.
8. The method of claim 1 , wherein the (−)-premnaspirodiene is isolated from the non-polar solvent phase by distillation.
9. The method of claim 8 , wherein the non-polar solvent phase is a vegetable oil.
10. The method of claim 9 , wherein the vegetable oil is a soybean oil.
11. The method of claim 8 , wherein the non-polar solvent phase is a high-molecular weight aliphatic hydrocarbon selected from among a straight-chain or branched-chain isomer of dodecane, tridecane, tetradecane, pentadecane, and hexadecane, wherein the high-molecular weight aliphatic hydrocarbon optionally is substituted with one or more hydroxy or halogen substituents such that the substituted hydrocarbon is substantially immiscible with the aqueous phase.
12. The method of claim 1 , wherein the premnaspirodiene synthase gene is from Hyoscyamus muticus.
13. The method of claim 5 , wherein the yeast cell overproduces farnesyl diphosphate.
14. The method of claim 1 , wherein the host cell is a bacteria, cyanobacteria, filamentous fungi or algae.
15. The method of claim 1 , wherein the culturing of the host cell is performed by batch fermentation, fed-batch fermentation or continuous fermentation.
16. The method of claim 7 , wherein sequestration is effected by binding the (−)-premnaspirodiene to a hydrophobic resin, and the (−)-premnaspirodiene is isolated from the resin.
17. The method of claim 7 , wherein culturing is effected by fermentation.