Production of isoprenoids
View Patent ↗The invention provides a biological method of producing isoprenoids.
1. A fermentation method comprising:
a. contacting a recombinant yeast with a fermentation medium wherein the recombinant yeast comprises a plurality of chromosomally integrated recombinant nucleic acid molecules, wherein each recombinant nucleic acid molecule encodes an enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid;
wherein the recombinant yeast is capable of producing an isoprenoid product having 5, 10, 15 or 20 carbons and wherein the fermentation medium comprises a sugar carbon source in a first amount between about 1 gram per liter and about 100 gram per liter of fermentation medium;
b. culturing the recombinant yeast under conditions wherein the sugar carbon source in the fermentation medium is consumed such that the fermentation medium comprises the sugar carbon source in a second amount which is less than the first amount;
c. maintaining the culturing conditions such that the fermentation medium comprises the sugar carbon source in an amount that is equal to or less than the second amount; and
d. recovering the product.
2. The method of claim 1 , wherein the first amount is between about 2 grams per liter and about 50 grams per liter of fermentation medium.
3. The method of claim 1 , wherein the first amount is between about 5 grams per liter and about 20 grams per liter of fermentation medium.
4. The method of claim 1 , wherein the second amount is about 1 gram per liter of fermentation medium.
5. The method of claim 1 , wherein the second amount is about 0.1 gram per liter of fermentation medium.
6. The method of claim 1 , wherein the second amount is undetectable.
7. The method of claim 1 , wherein the culturing conditions are maintained by making additions of the carbon source to the fermentation medium as the carbon source is depleted.
8. The method of claim 1 , wherein the culturing conditions are maintained between about 28° C. and about 32° C.
9. A fermentation method, comprising:
a. culturing a recombinant yeast that is capable of producing an isoprenoid product having 5, 10, 15 or 20 carbons, comprising a plurality of chromosomally integrated recombinant nucleic acid molecules, wherein each recombinant nucleic acid molecule encodes an enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid in a fermentation medium under fed-batch conditions until the microorganism reaches a cell density of at least 10 grams of dry cell weight per liter of fermentation medium; and
b. recovering the isoprenoid product.
10. The method of claim 9 , wherein the culturing step is under carbon source restricted conditions.
11. The method of claim 10 , wherein the carbon source is maintained at a concentration less than about 20 grams per liter of fermentation medium.
12. The method of claim 10 , wherein the carbon source is maintained at a concentration less than about 0.1 gram per liter of fermentation medium.
13. The method of claim 10 , wherein the carbon source is maintained at undetectable levels.
14. The method of claim 10 , wherein the carbon source is selected from the group consisting of monosaccharides, disaccharides, and trisaccharides.
15. The method of claim 10 , wherein the carbon source is glucose.
16. The method of claim 9 , wherein the culturing step is conducted under nitrogen source restricted conditions.
17. The method of claim 16 , wherein the concentration of assimilable nitrogen is maintained at less than about 20 g/L in the fermentation medium.
18. The method of claim 16 , wherein the concentration of assimilable nitrogen is maintained at less than about 10 g/L in the fermentation medium.
19. The method of claim 16 , wherein the concentration of assimilable nitrogen is maintained at less than about 5 g/L in the fermentation medium.
20. The method of claim 16 , wherein the concentration of assimilable nitrogen is maintained at undetectable levels.
21. The method of claim 16 , wherein the nitrogen source is selected from the group consisting of anhydrous ammonia and ammonium salts.
22. The method of claim 16 , wherein the nitrogen source is selected from the group consisting of protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids.
23. The method of claim 9 , wherein the culturing step is conducted under both carbon source and nitrogen source restricted conditions.
24. The method of claim 9 , wherein the culturing conditions are maintained between about 20° C. and about 45° C.
25. The method of claim 9 , wherein the cell density is between about 20 grams of dry cell weight per liter of fermentation medium and about 80 grams of dry cell weight per liter of fermentation medium prior to recovering the product.
26. The method of claim 9 , wherein the cell density is between about 50 grams of dry cell weight per liter of fermentation medium and about 70 grams of dry cell weight per liter of fermentation medium prior to recovering the product.
27. The method of claim 9 , wherein the microorganism is Saccharomyces cerevisiae.
28. The method of claim 1 , wherein the yeast is Saccharomyces cerevisiae.
29. The method of claim 1 , wherein the isoprenoid product is a 5 carbon compound.
30. The method of claim 1 , wherein the isoprenoid product is a 10 carbon compound.
31. The method of claim 1 , wherein the isoprenoid product is a 15 carbon compound.
32. The method of claim 1 , wherein the isoprenoid product is a 20 carbon compound.
33. The method of claim 1 , wherein the culturing conditions are maintained between about 20° C. and about 45° C.
34. The method of claim 1 , wherein the culturing conditions are maintained between about 25° C. and about 40° C.
35. The method of claim 1 , wherein the enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid is a bacterial enzyme.
36. The method of claim 1 , wherein the enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid is a yeast enzyme.
37. The method of claim 1 , wherein the sugar carbon source is selected from the group consisting of monosaccharides, disaccharides, and trisaccharides.