Production of isoprenoids

Abstract

A recombinant yeast that makes an isoprenoid compound is provided. The yeast comprises an endogenous mevalonate pathway comprising (i) an enzyme that converts acetyl-CoA to acetoacetyl-CoA, (ii) an enzyme that converts acetoacetyl-CoA to hydroxymethylglutaryl-CoA, (iii) an enzyme that converts hydroxymethylglutaryl-CoA to mevalonic acid, (iv) an enzyme that converts mevalonic acid to phosphomevalonic acid, (v) an enzyme that converts phosphomevalonic acid to phosphomevalonate, and (vi) an enzyme that converts phosphomevalonate to isopentenyl pyrophosphate. The yeast further comprises heterologous nucleic acid sequences encoding an enzyme that converts hydroxymethylglutaryl-CoA to mevalonic acid, an enzyme that converts mevalonic acid to phosphomevalonic acid, an enzyme that converts phosphomevalonic acid to phosphomevalonate, and an enzyme that converts phosphomevalonate to isopentenyl pyrophosphate. Carbon flow through the mevalonate pathway is increased in the recombinant yeast compared to a yeast that does not comprise the heterologous nucleic acid molecule encoding an enzyme that converts hydroxymethylglutaryl-CoA to mevalonic acid.

What is claimed is: 
     
         1 . A fermentation method comprising:
 (a) contacting a recombinant microorganism with a fermentation medium wherein the recombinant microorganism comprises one or more chromosomally integrated recombinant nucleic acid molecules encoding an enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid,
 wherein the recombinant microorganism 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 an amount between about 1 gram per liter and about 100 grams per liter of the fermentation medium; 
   (b) culturing the recombinant microorganism under conditions wherein the sugar carbon source in the fermentation medium is consumed; and   (c) recovering the product.   
     
     
         2 . The method of  claim 1 , wherein the culturing step is performed under carbon restricted conditions. 
     
     
         3 . The method of  claim 1 , wherein the carbon source is maintained at a concentration less than about 20 grams per liter of the fermentation medium. 
     
     
         4 . The method of  claim 1 , further comprising:
 (a) monitoring the concentration of the sugar carbon source in the fermentation medium; and   (b) making additions of the sugar carbon source to the fermentation medium as the sugar carbon source is depleted.   
     
     
         5 . The method of  claim 1 , wherein the culturing conditions are maintained between about 25° C. and about 40° C. 
     
     
         6 . The method of  claim 1 , wherein the pH of the fermentation medium is maintained from about 4.0 to about 6.5. 
     
     
         7 . The method of  claim 1 , wherein the sugar carbon source is selected from the group consisting of monosaccharides, disaccharides, and trisaccharides. 
     
     
         8 . The method of  claim 1 , wherein the microorganism is cultured 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. 
     
     
         9 . The method of  claim 8 , wherein the cell density is between about 20 grams of dry cell weight per liter of the fermentation medium and about 80 grams of dry cell weight per liter of the fermentation medium prior to recovering the product. 
     
     
         10 . The method of  claim 1 , wherein the enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid is a bacterial enzyme. 
     
     
         11 . The method of  claim 1 , wherein the enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid is a yeast enzyme. 
     
     
         12 . The method of  claim 1 , wherein the recombinant microorganism comprises multiple copies of chromosomally integrated recombinant nucleic acid molecules encoding the enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid. 
     
     
         13 . The method of  claim 1 , wherein the enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid is an HMG Co A reductase. 
     
     
         14 . The method of  claim 1 , wherein the microorganism further comprises at least one additional chromosomally integrated recombinant nucleic acid molecule encoding one or more enzymes to produce the isoprenoid product. 
     
     
         15 . The method of  claim 1 , wherein the isoprenoid product is a 5 carbon compound. 
     
     
         16 . The method of  claim 1 , wherein the isoprenoid product is a 10 carbon compound. 
     
     
         17 . The method of  claim 1 , wherein the isoprenoid product is a 15 carbon compound. 
     
     
         18 . The method of  claim 1 , wherein the isoprenoid product is a 20 carbon compound. 
     
     
         19 . The method of  claim 1 , wherein the microorganism is a fungus. 
     
     
         20 . The method of  claim 1 , wherein the microorganism is a yeast. 
     
     
         21 . The method of  claim 1 , wherein the microorganism is  Saccharomyces cerevisiae.    
     
     
         22 . The method of  claim 1 , wherein the microorganism is a bacterium. 
     
     
         23 . A recombinant microorganism comprising:
 (a) one or more chromosomally integrated recombinant nucleic acid molecules encoding an enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid, and   (b) at least one additional chromosomally integrated recombinant nucleic acid molecule encoding one or more enzymes in an isoprenoid pathway to produce an isoprenoid product having 5, 10, 15, or 20 carbons.   
     
     
         24 . A fermentation composition comprising:
 (a) a recombinant microorganism in a fermentation medium, the recombinant microorganism comprising:
 (i) one or more chromosomally integrated recombinant nucleic acid molecules encoding an enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid, and 
 (ii) at least one additional chromosomally integrated recombinant nucleic acid molecule encoding one or more enzymes in an isoprenoid pathway to produce an isoprenoid product having 5, 10, 15, or 20 carbons; and 
   (b) the isoprenoid product having 5, 10, 15, or 20 carbons.   
     
     
         25 . The fermentation composition of  claim 24 , wherein the enzyme that converts hydroxymethylglutaryl-CoA to L-mevalonic acid is an HMG Co A reductase. 
     
     
         26 . The fermentation composition of  claim 24 , wherein the isoprenoid product is a 5 carbon compound. 
     
     
         27 . The fermentation composition of  claim 24 , wherein the isoprenoid product is a 10 carbon compound. 
     
     
         28 . The fermentation composition of  claim 24 , wherein the isoprenoid product is a 15 carbon compound. 
     
     
         29 . The fermentation composition of  claim 24 , wherein the isoprenoid product is a 20 carbon compound. 
     
     
         30 . The fermentation composition of  claim 24 , wherein the microorganism is a fungus. 
     
     
         31 . The fermentation composition of  claim 24 , wherein the microorganism is a yeast. 
     
     
         32 . The fermentation composition of  claim 24 , wherein the microorganism is  Saccharomyces cerevisiae.    
     
     
         33 . The fermentation composition of  claim 24 , wherein the microorganism is a bacterium