Host cells and methods for producing 1-deoxyxylulose 5-phosphate (DXP) and/or a DXP derived compound

Patent metadata

Field	Value
Publication number	US-9382553-B2
Application number	US-201213587826-A
Country	US
Kind code	B2
Filing date	Aug 16, 2012
Priority date	Aug 16, 2011
Publication date	Jul 5, 2016
Grant date	Jul 5, 2016

Abstract

The present invention provides for a genetically modified host cell capable of producing 1-deoxyxylulose 5-phosphate or 1-deoxy-D-xylulose 5-phosphate (DXP) (12), and optionally one or more DXP derived compounds, comprising: (a) a mutant RibB, or functional variant thereof, capable of catalyzing xylulose 5-phosphate and/or ribulose 5-phosphate to DXP, or (b) a YajO, or functional variant thereof, and a XylB, or functional variant thereof.

First claim

We claim:

1. A genetically modified host cell capable of producing 1-deoxyxylulose 5-phosphate or 1-deoxy-D-xylulose 5-phosphate (DXP) comprising: a mutant 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) comprising a polypeptide sequence that is identical to SEQ ID NO:1 except the polypeptide sequence comprises one or more of the following amino acid substitutions corresponding to substitutions in SEQ ID NO:1 selected from the group consisting of G108S, T88I, S89R, V109I, M182I, G92D, and T106I; wherein the host cell is a prokaryotic cell.

2. The host cell of claim 1 , wherein the host cell does not naturally comprise a gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase (dxs).

3. The host cell of claim 1 , wherein the host cell (a) further comprises a disruption in an endogenous gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase, or (b) further comprises a mutation in an endogenous gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase which reduces 1-deoxy-D-xylulose 5-phosphate synthase activity or reduces expression of said gene.

4. The host cell of claim 1 , wherein the host cell is capable of producing 2C-methyl-D-erythritol 4-phosphate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase.

5. The host cell of claim 1 , wherein the host cell is capable of producing 4-diphosphocytidyl-2C-methyl-D-erythritol, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase and a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase.

6. The host cell of claim 1 , wherein the host cell is capable of producing 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, and a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase.

7. The host cell of claim 1 , wherein the host cell is capable of producing 2C-methyl-D-erythritol 2,4-diphosphate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, and a 2C-methyl-D-erythritol 2,4-diphosphate synthase.

8. The host cell of claim 1 , wherein the host cell is capable of producing 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, a 2C-methyl-D-erythritol 2,4-diphosphate synthase, and a 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase.

9. The host cell of claim 1 , wherein the host cell is capable of producing isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP), and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, a 2C-methyl-D-erythritol 2,4-diphosphate synthase, a 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase, and a 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase.

10. The host cell of claim 1 , wherein the host cell is capable of producing geranyl pyrophosphate (GPP) and optionally farnesyl pyrophosphate (FPP), and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, a 2C-methyl-D-erythritol 2,4-diphosphate synthase, a 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase, a 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, a GPP synthase and optionally a FPP synthase.

11. The host cell of claim 1 , wherein the host cell is capable of producing DXP derivatives, wherein said DXP derivatives are (i) isopentenol, geraniol, and optionally farnesol, or (ii) an isoprenyl alkanoate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, a 2C-methyl-D-erythritol 2,4-diphosphate synthase, a 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase, a 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, a GPP synthase, the corresponding enzymes for producing the DXP derivatives of (i) or (ii), and optionally a FPP synthase.

12. A genetically modified host cell capable of producing 1-deoxyxylulose 5-phosphate or 1-deoxy-D-xylulose 5-phosphate (DXP) comprising (a) an aldo/keto reductase (YajO) having the amino acid sequence of SEQ ID NO: 2, and (b) a xylulokinase (xylB) having the amino acid sequence of SEQ ID NO: 3, wherein the host cell (i) does not naturally comprise a gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase, or (ii) further comprises a disruption in an endogenous gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase, wherein said host cell is a prokaryotic cell, and wherein:
(I) the host cell is capable of producing GPP and optionally FPP, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, a 2C-methyl-D-erythritol 2,4-diphosphate synthase, a 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase, a 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, a GPP synthase and optionally a FPP synthase; or
(II) the host cell is capable of producing DXP derivatives, wherein said DXP derivatives are (i) isopentenol, geraniol, and optionally farnesol, or (ii) an isoprenyl alkanoate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, a 2C-methyl-D-erythritol 2,4-diphosphate synthase, a 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase, a 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, a GPP synthase, the corresponding enzymes for producing the DXP derivatives of (i) or (ii), and optionally a FPP synthase.

13. The host cell of claim 12 , wherein the host cell does not naturally comprise a gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase.

14. The host cell of claim 12 , wherein the host cell further comprises a disruption in an endogenous gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase.

15. The host cell of claim 12 , wherein the host cell is capable of producing 2C-methyl-D-erythritol 4-phosphate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase.

16. The host cell of claim 12 , wherein the host cell is capable of producing 4-diphosphocytidyl-2C-methyl-D-erythritol, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase and a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase.

17. The host cell of claim 12 , wherein the host cell is capable of producing 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, and a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase.

18. The host cell of claim 12 , wherein the host cell is capable of producing 2C-methyl-D-erythritol 2,4-diphosphate, and the host cell further comprises a 2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol 4-phosphate synthase, a 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, and a 2C-methyl-D-erythr

Assignees

• Kirby James
• Fortman Jeffrey L
• Nishimoto Minobu
• Keasling Jay D
• Univ California

Inventors

• Kirby James
• Fortman Jeffrey L
• Nishimoto Minobu
• Keasling Jay D

Classifications

• C12P5/007 Primary

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title

• C12P19/02 Primary

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title