Methods for microbial production of terpenoids

Abstract

The invention relates to recombinant expression of terpenoid synthase enzymes and geranylgeranyl diphosphate synthase (GGPPS) enzymes in cells and the production of diterpenoids.

1 . A method comprising:
 recombinantly expressing a terpenoid synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in a cell that overexpresses one or more components of the non-mevalonate (MEP) pathway.   
     
     
         2 . The method of  claim 1 , wherein the cell is a bacterial cell, a yeast cell, an algal cell or a plant cell. 
     
     
         3 .- 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the terpenoid synthase enzyme is a diterpenoid synthase enzyme, optionally wherein the diterpenoid synthase enzyme is a levopimaradiene synthase (LPS) enzyme, optionally wherein the LPS enzyme contains one or more mutations. 
     
     
         12 .- 14 . (canceled) 
     
     
         15 . The method of  claim 11 , wherein the LPS enzyme contains a mutation at one or more of the residues selected from the group consisting of: M593, C618, A620, L696, Y700, K723, A729, V731, N838, and I855, corresponding to residues within the full-length, wild-type,  Ginkgo biloba  LPS enzyme, or one or more mutations in equivalent residues within a homologous LPS enzyme, optionally wherein the LPS enzyme contains one or more mutations selected from the group consisting of: M593I, M593L, C618N, L696Q, K723S, V731L, N838E, 1855L, A729G, Y700H, Y700A, Y700C, Y700F, Y700M, Y700W, A620C, A620G, A620S, A620T and A620V, corresponding to mutations at residues within the full-length, wild-type,  Ginkgo biloba  LPS enzyme, or one or more equivalent mutations in a homologous LPS enzyme. 
     
     
         16 .- 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the GGPPS enzyme contains one or more mutations. 
     
     
         20 . The method of  claim 19 , wherein the GGPPS enzyme contains a mutation at residue S239 and/or G295, corresponding to residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or a mutation in one or both equivalent residues within a homologous GGPPS enzyme, optionally wherein the GGPPS enzyme contains the mutation S239C and/or G295D, corresponding to mutations at residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or one or both equivalent mutations in a homologous GGPPS enzyme. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 11 , wherein the LPS enzyme contains the mutation M593I and/or Y700F, corresponding to residues within the full-length wild-type  Ginkgo biloba  LPS enzyme, or one or more equivalent mutations in a homologous LPS enzyme, and the GGPPS enzyme contains the mutation S239C and/or G295D, corresponding to residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or one or more equivalent mutations in a homologous GGPPS enzyme. 
     
     
         23 .- 24 . (canceled) 
     
     
         25 . The method of  claim 1 , further comprising culturing the cell. 
     
     
         26 . The method of  claim 1 , wherein the cell produces a terpenoid, optionally wherein the terpenoid has one or more cyclic structures. 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 26 , wherein the terpenoid is a diterpenoid, optionally wherein the diterpenoid is levopimaradiene, a Taxol, a gibberellin, and/or a steviol glycoside. 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 25 , further comprising recovering the terpenoid from the cell culture. 
     
     
         31 .- 34 . (canceled) 
     
     
         35 . A cell that overexpresses one or more components of the non-mevalonate (MEP) pathway, and that recombinantly expresses a terpenoid synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme. 
     
     
         36 . The cell of  claim 35 , wherein the cell is a bacterial cell, a yeast cell, an algal cell or a plant cell. 
     
     
         37 .- 44 . (canceled) 
     
     
         45 . The cell of  claim 35 , wherein the terpenoid synthase enzyme is a diterpenoid synthase enzyme, optionally wherein the diterpenoid synthase enzyme is a levopimaradiene synthase (LPS) enzyme, optionally wherein the LPS enzyme contains one or more mutations. 
     
     
         46 .- 48 . (canceled) 
     
     
         49 . The cell of  claim 45 , wherein the LPS enzyme contains a mutation at one or more of the residues selected from the group consisting of: M593, C618, A620, L696, Y700, K723, A729, V731, N838, and I855, corresponding to residues within the full-length, wild-type,  Ginkgo biloba  LPS enzyme, or one or more mutations in equivalent residues within a homologous LPS enzyme, optionally wherein the LPS enzyme contains one or more mutations selected from the group consisting of: M593I, M593L, C618N, L696Q, K723S, V731L, N838E, I855L, A729G, Y700H, Y700A, Y700C, Y700F, Y700M, Y700W, A620C, A620G, A620S, A620T and A620V, corresponding to mutations at residues within the full-length, wild-type,  Ginkgo biloba  LPS enzyme, or one or more equivalent mutations in a homologous LPS enzyme. 
     
     
         50 .- 52 . (canceled) 
     
     
         53 . The cell of  claim 35 , wherein the GGPPS enzyme contains one or more mutations. 
     
     
         54 . The cell of  claim 53 , wherein the GGPPS enzyme contains a mutation at residue S239 and/or G295, corresponding to residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or a mutation in one or both equivalent residues within a homologous GGPPS enzyme, optionally wherein the GGPPS enzyme contains the mutation S239C and/or G295D, corresponding to mutations at residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or one or both equivalent mutations in a homologous GGPPS enzyme. 
     
     
         55 . (canceled) 
     
     
         56 . The cell of  claim 45 , wherein the LPS enzyme contains the mutation M593I and/or Y700F, corresponding to residues within the full-length wild-type  Ginkgo biloba  LPS enzyme, or one or more equivalent mutations in a homologous LPS enzyme, and the GGPPS enzyme contains the mutation S239C and/or G295D, corresponding to residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or one or more equivalent mutations in a homologous GGPPS enzyme. 
     
     
         57 .- 58 . (canceled) 
     
     
         59 . The cell of  claim 35 , wherein the cell produces a terpenoid, optionally wherein the terpenoid has one or more cyclic structures. 
     
     
         60 . (canceled) 
     
     
         61 . The cell of  claim 59 , wherein the terpenoid is a diterpenoid optionally wherein the diterpenoid is levopimaradiene, a Taxol, a gibberellin, and/or a steviol glycoside. 
     
     
         62 .- 64 . (canceled) 
     
     
         65 . A cell that recombinantly expresses a levopimaradiene synthase (LPS) enzyme, wherein the LPS enzyme contains a mutation at one or more of the residues selected from the group consisting of: M593, C618, A620, L696, Y700, K723, A729, V731, N838, and I855, corresponding to residues within the full-length, wild-type,  Ginkgo biloba  LPS enzyme, or one or more mutations in equivalent residues within a homologous LPS enzyme, optionally wherein the LPS enzyme contains one or more mutations selected from the group consisting of: M593I, M593L, C618N, L696Q, K723S, V731L, N838E, 1855L, A729G, Y700H, Y700A, Y700C, Y700F, Y700M, Y700W, A620C, A620G, A620S, A620T and A620V, corresponding to mutations at residues within the full-length, wild-type,  Ginkgo biloba  LPS enzyme, or one or more equivalent mutations in a homologous LPS enzyme, optionally wherein the LPS enzyme contains the mutation M593I and one of the mutations selected from the group consisting of Y700A, Y700C and Y700F, corresponding to mutations at residues within the full-length, wild-type,  Ginkgo biloba  LPS enzyme, or one or more equivalent mutations in a homologous LPS enzyme. 
     
     
         66 .- 77 . (canceled) 
     
     
         78 . A cell that recombinantly expresses a geranylgeranyl diphosphate synthase (GGPPS) enzyme, wherein the GGPPS enzyme contains a mutation at residue S239 and/or G295, corresponding to residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or a mutation in one or both equivalent residues within a homologous GGPPS enzyme optionally wherein the GGPPS enzyme contains the mutation S239C and/or G295D, corresponding to mutations at residues within the full-length, wild-type,  Taxus canadensis  GGPPS enzyme, or one or both equivalent mutations in a homologous GGPPS enzyme. 
     
     
         79 .- 104 . (canceled)