PRODUCTION OF ISOPRENOIDS
The present invention provides methods for a robust production of isoprenoids utilizing the DXP biosynthetic path-way. The invention also provides nucleic acids, enzymes, expression vectors, and genetically modified host cells for carrying out the subject methods.
1 . A genetically modified host cell capable of converting pyruvate and glyceralderhyde 3-phosphate into dimethylallyldiphosphate (DMAPP), wherein the host cell comprises heterologous nucleic acid sequences that encode:
a. an enzyme that converts pyruvate and glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose-5-phosphate (DXP);
b. an enzyme that converts 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP) to IPP, and,
c. an enzyme that converts IPP to DMAPP.
2 . The host cell of claim 1 wherein the host cell additionally comprises one or more additional heterologous nucleic acid sequences that encode one or more enzymes selected from the group consisting of:
a. an enzyme that converts DXP to 2C-methyl-D-erythritol-4-phosphate (MEP);
b. an enzyme that converts MEP to 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME);
c. an enzyme that converts CDP-ME to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate (CDP-MEP);
d. an enzyme that converts CDP-MEP to 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC); and
e. an enzyme that converts MEC to HMBPP.
3 . The host cell of claim 1 wherein the host cell comprises a heterologous nucleic acid sequence that encodes an enzyme that converts IPP and DMAPP into geranyl pyrophosphate.
4 . The host cell of claim 1 wherein the host cell comprises a heterologous nucleic acid sequence that encodes an enzyme that converts IPP and DMAPP into farnesyl pyrophosphate.
5 . The host cell of claim 1 wherein the host cell comprises a heterologous nucleic acid sequence that encodes an enzyme that converts IPP and DMAPP into geranylgeranyl pyrophosphate.
6 . The host cell of claim 1 , wherein the host cell has an endogenous MEV pathway.
7 . The host cell of claim 4 wherein the host cell is S. cerevisiae.
8 . The host cell of claim 1 , wherein the host cell has an endogenous DXP pathway.
9 . The host cell of claim 6 wherein the host cell is E. coli.
10 . A genetically modified host cell having an endogenous DXP pathway comprising heterologous nucleic acid sequences wherein the heterologous nucleic acid sequences encode:
a. an enzyme that converts pyruvate and glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose-5-phosphate (DXP);
b. an enzyme that converts DXP to 2C-methyl-D-erythritol-4-phosphate (MEP);
c. an enzyme that converts MEP to 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME);
d. an enzyme that converts CDP-ME to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate (CDP-MEP);
e. an enzyme that converts CDP-MEP to 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC);
f. an enzyme that converts MEC to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP);
g. an enzyme that converts HMBPP to IPP, and,
h. an enzyme that converts IPP to DMAPP.
11 . The host cell of claim 10 wherein the heterologous nucleic acid sequences are under the control of a single regulatory element.
12 . The host cell of claim 10 wherein the host cell comprises a heterologous nucleic acid sequence that encodes an enzyme that converts IPP and DMAPP into geranyl pyrophosphate.
13 . The host cell of claim 10 wherein the host cell comprises a heterologous nucleic acid sequence that encodes an enzyme that converts IPP and DMAPP into farnesyl pyrophosphate.
14 . The host cell of claim 10 wherein the host cell comprises a heterologous nucleic acid sequence that encodes an enzyme that converts IPP and DMAPP into geranylgeranyl pyrophosphate.
15 . The host cell of claim 10 wherein the host cell is E. coli.
16 . A method for making an isoprenoid or isoprenoid precursor, comprising: culturing in an aqueous medium a genetically modified host cell capable of converting pyruvate and glyceralderhyde 3-phosphate into dimethylallyldiphosphate (DMAPP), wherein the host cell comprises heterologous nucleic acid sequences that encode:
a. an enzyme that converts pyruvate and glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose-5-phosphate (DXP);
b. an enzyme that converts 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP) to IPP, and,
c. an enzyme that converts IPP to DMAPP.
17 . The method of claim 16 wherein the host cell comprises one or more additional heterologous nucleic acid sequences that encode one or more enzymes selected from the group consisting of:
a. an enzyme that converts DXP to 2C-methyl-D-erythritol-4-phosphate (MEP);
b. an enzyme that converts MEP to 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME);
c. an enzyme that converts CDP-MEP to 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEG); and
d. an enzyme that converts MEG to HMBPP.
18 . A method of making an isoprenoid or an isoprenoid precursor, comprising culturing in an aqueous medium a genetically modified host cell having an endogenous DXP pathway, wherein the host cell comprises heterologous nucleic acid sequences that encode:
a. an enzyme that converts pyruvate and glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose-5-phosphate (DXP);
b. an enzyme that converts DXP to 2C-methyl-D-erythritol-4-phosphate (MEP);
c. an enzyme that converts MEP to 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME);
d. an enzyme that converts CDP-ME to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate (CDP-MEP);
e. an enzyme that converts CDP-MEP to 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC);
f. an enzyme that converts MEC to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP);
g. an enzyme that converts HMBPP to IPP, and,
h. an enzyme that converts IPP to DMAPP.
19 . The method of claim 18 wherein the heterologous nucleic acid sequences are under the control of a single regulatory element.
20 . The method of claim 18 wherein the host cell is E. coli.