Production of isoprenoids

Abstract

The present invention provides methods for a robust production of isoprenoids via one or more biosynthetic pathways. The invention also provides nucleic acids, enzymes, expression vectors, and genetically modified host cells for carrying out the subject methods. The invention also provides fermentation methods for high productivity of isoprenoids from genetically modified host cells.

1 - 16 . (canceled) 
     
     
         17 . A method for producing an isoprenoid comprising:
 culturing a microorganism in a medium with a limited carbon source that provides for 75% or less of a maximum specific growth rate of the microorganism, wherein the maximum specific growth rate is a rate that would have been achieved by culturing the microorganism at an optimal temperature for growth in a medium in which nutrients are present in excess,   wherein the microorganism comprises a heterologous nucleic acid encoding one or more enzymes of a deoxyxylulose 5-phosphate (DXP) pathway for making isopentenyl pyrophosphate, under control of at least one heterologous transcriptional regulator, and   wherein the heterologous nucleic acid encodes at least one enzyme selected from the group consisting of:   (i) an enzyme that condenses pyruvate with D-glyceraldehyde 3-phosphate to form 1-deoxy-D-xylulose-5-phosphate;   (ii) an enzyme that converts 1-deoxy-D-xylulose-5-phosphate to 2C-methyl-D-erythritol-4-phosphate;   (iii) an enzyme that converts 2C-methyl-D-erythritol-4-phosphate to 4-diphosphocytidyl-2C-methyl-D-erythritol;   (iv) an enzyme that converts 4-diphosphocytidyl-2C-methyl-D-erythritol to 4-diphosphocytidyl-2C-methyl-D-erythrito1-2-phosphate;   (v) an enzyme that converts 4-diphosphocytidyl-2C-methyl-D-erythrito1-2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate;   (vi) an enzyme that converts 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate; and,   (vii) an enzyme that converts 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate to one of IPP or DMAPP.   
     
     
         18 . The method of  claim 17  wherein the at least one heterologous transcriptional regulator is inducible. 
     
     
         19 . The method of  claim 17  wherein the DXP pathway enzyme is under control of a single transcriptional regulator. 
     
     
         20 . The method of  claim 17  wherein the DXP pathway enzyme is under control of multiple transcriptional regulators. 
     
     
         21 . The method of  claim 17  wherein the microorganism comprises a plurality of heterologous nucleic acids encoding all of the enzymes of a DXP pathway. 
     
     
         22 . The method of  claim 17  wherein the microorganism is  E. coli.    
     
     
         23 . The method of  claim 17  wherein the microorganism is  S. cerevisiae.    
     
     
         24 . The method of  claim 17  wherein the heterologous nucleic acid comprises a nucleic acid sequence encoding a DXP pathway enzyme from a bacterium having an endogenous DXP pathway. 
     
     
         25 . The method of  claim 24  wherein the bacterium is of a genus selected from  Enterococcus, Pseudomonas,  and  Staphyloccoccus.    
     
     
         26 . The method of  claim 17  wherein the heterologous nucleic acid comprises a nucleic acid sequence encoding a DXP pathway enzyme selected from 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, and 4-diphosphocytidyl-2C-methyl-D-erythritol synthase. 
     
     
         27 . The method of  claim 17  wherein the heterologous nucleic acid comprises a nucleic acid sequence encoding a DXP pathway enzyme selected from 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, and isopentyl/dimethylallyl diphosphate synthase. 
     
     
         28 . The method of  claim 17  wherein the microorganism is cultured in a growth medium having both nutrients and temperatures maintained at levels below those that provide for the maximum specific growth rate of the host cells. 
     
     
         29 . The method of  claim 17  wherein the temperature of the medium is at least 2-20° C. below the optimal temperature. 
     
     
         30 . The method of  claim 29  wherein the microorganism is cultured at a temperature at least 5° C. below the optimal temperature. 
     
     
         31 . The method of  claim 29  wherein the temperature of the medium is at least 10° C. below the optimal temperature. 
     
     
         32 . The method of  claim 17  wherein the medium provides for about 60% or less of the maximum specific growth rate. 
     
     
         33 . The method of  claim 17  wherein the medium provides for about 50% or less of the maximum specific growth rate. 
     
     
         34 . The method of  claim 17  wherein the medium provides for about 40% or less of the maximum specific growth rate. 
     
     
         35 . The method of  claim 17  wherein the medium provides for about 25% or less of the maximum specific growth rate. 
     
     
         36 . The method of  claim 17  wherein the medium provides for about 75%-10% of the maximum specific growth rate. 
     
     
         37 . The method of  claim 17  wherein the medium is nitrogen-restricted. 
     
     
         38 . The method of  claim 17  wherein the isoprenoid is produced in an amount greater than about 10 grams per liter of medium. 
     
     
         39 . The method of  claim 17  wherein the isoprenoid is produced in an amount greater than about 50 mg per gram of dry cell weight. 
     
     
         40 . The method of  claim 38  or  39  wherein the amount of isoprenoid is produced in less than about 150 hours. 
     
     
         41 . The method of  claim 38  or  39  wherein the amount of isoprenoid is produced in less than about 96 hours. 
     
     
         42 . The method of  claim 38  or  39  wherein the amount of isoprenoid is produced in less than about 72 hours. 
     
     
         43 . The method of  claim 17  wherein the isoprenoid is selected from the group consisting of a hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, sesquiterpene, and polyterpene. 
     
     
         44 . The method of  claim 17  wherein the isoprenoid is a sesquiterpene. 
     
     
         45 . The method of  claim 17  wherein the isoprenoid is a C 5 -C 20  isoprenoid. 
     
     
         46 . The method of  claim 17 , wherein the nutrients comprise a carbon source and a nitrogen source. 
     
     
         47 . The method of  claim 17 , wherein said one or more enzymes is a bacterial enzyme. 
     
     
         48 . The method of  claim 17 , wherein said one or more enzymes is an  E. coli  enzyme. 
     
     
         49 . The method of  claim 17  wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-famesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpindene and valencene. 
     
     
         50 . The method of  claim 49  wherein the sesquiterpene is α-farnesene. 
     
     
         51 . The method of  claim 49  wherein the sesquiterpene is β-farnesene. 
     
     
         52 . The method of  claim 49  wherein the sesquiterpene is amorphadiene. 
     
     
         53 . The method of  claim 49  wherein the sesquiterpene is farnesol. 
     
     
         54 . The method of  claim 49  wherein the sesquiterpene is nerolidol. 
     
     
         55 . The method of  claim 49  wherein the sesquiterpene is patchoulol. 
     
     
         56 . The method of  claim 49  wherein the sesquiterpene is valencene.