Methods for increasing production of 3-methyl-2-butenol using fusion proteins

Patent metadata

Field	Value
Publication number	US-9631210-B2
Application number	US-201214345147-A
Country	US
Kind code	B2
Filing date	Sep 13, 2012
Priority date	Sep 14, 2011
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

Abstract

The present invention relates to methods and compositions for increasing production of 3-methyl-2-butenol in a de novo synthetic pathway in a genetically modified host cell using isopentenyl disphosphate (IPP) as a substrate.

First claim

What is claimed is:

1. A genetically modified host cell transformed with a nucleic acid construct encoding a fusion protein comprising a phosphatase capable of catalyzing the dephosphorylation of dimethylallyl diphosphate (DMAPP) linked to an isopentyl pyrophosphate (IPP) isomerase capable of converting IPP to DMAPP, wherein the nucleic acid construct is operably linked to a promoter.

2. The genetically modified host cell of claim 1 , wherein the genetically modified host cell further comprises a nucleic acid encoding a reductase that is capable of converting 3-methyl-2-butenol to 3-methy-butanol.

3. The genetically modified host cell of claim 2 , wherein the reductase is encoded by a nucleic acid construct introduced into the cell.

4. The genetically modified host cell of claim 1 , wherein the IPP isomerase is a Type I isomerase.

5. The genetically modified host cell of claim 4 , wherein the IPP isomerase is encoded by the E. coli idi gene or the Saccharomyces cerevisiae idil gene.

6. The genetically modified host cell of claim 1 , wherein the phosphatase is a member of the Nudix superfamily.

7. The genetically modified host cell of claim 6 , wherein the phosphatase is encoded by the E. coli nudB gene.

8. The genetically modified host cell of claim 1 , wherein the host cell is a prokaryotic cell selected from Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia , Vitreoscilla, Synechococcus, Synechocystis , or Paracoccus taxonomical classes.

9. The genetically modified host cell of claim 8 , wherein the prokaryotic cell is an Escherichia coli cell.

10. The genetically modified host cell of claim 1 , wherein the host cell is a fungal cell.

11. The genetically modified host cell of claim 10 , wherein the fungal cell is a yeast cell.

12. The genetically modified host cell of claim 11 , wherein the yeast cell is a Saccharomyces sp. cell.

13. The genetically modified host cell of claim 1 , wherein the host cell is an algal, insect or mammalian cell line.

14. The genetically modified host cell of claim 1 , wherein the IPP isomerase is encoded by the E. coli idi gene or the Saccharomyces cerevisiae idil gene; and the phosphatase is encoded by the E. coli nudB gene.

15. The genetically modified host cell of claim 14 , wherein the host cell is a prokaryotic cell.

16. The genetically modified host cell of claim 15 , wherein the host cell is a prokaryotic cell selected from Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis , or Paracoccus taxonomical classes.

17. The genetically modified host cell of claim 15 , wherein the prokaryotic cell is an Escherichia coli cell.

18. A method of enhancing production of a 3-methyl-2-butenol, the method comprising culturing a genetically modified host cell transformed with a nucleic acid construct encoding a fusion protein comprising a phosphatase capable of catalyzing the dephosphorylation of DMAPP linked to IPP isomerase capable of converting IPP to DMAPP, wherein the nucleic acid construct is operably linked to a promoter, under conditions such that the culturing results in the expression of the fusion protein and production of 3-methyl-2-butenol.

19. The method of claim 18 , further comprising recovering 3-methyl-2-butenol produced by the genetically modified host cell.

20. The method of claim 18 , wherein the genetically modified host cell further comprises a nucleic acid encoding a reductase such that expression of the reductase converts 3-methyl-2-butenol to 3-methyl butanol.

21. The method of claim 20 , wherein the reductase is encoded by a nucleic acid construct introduced into the genetically modified host cell.

22. The method of claim 20 , further comprising recovering 3-methyl-2-butenol or 3-methyl butanol produced by the genetically modified host cell.

23. The method of claim 18 , wherein the IPP isomerase is a Type I isomerase.

24. The method of claim 23 , wherein the IPP isomerase is encoded by the E. coli idi gene or the Saccharomyces cerevisiae idil gene.

25. The method of claim 18 , wherein the phosphatase is a member of the Nudix superfamily.

26. The method of claim 25 , wherein the phosphatase is encoded by the E. coli nudB gene.

27. The method of claim 18 , wherein the genetically modified host cell is a prokaryotic cell selected from Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis , or Paracoccus taxonomical classes.

28. The method of claim 27 , wherein the prokaryotic cell is an Escherichia coli cell.

29. The method of claim 18 , wherein the genetically modified host cell is a fungal cell.

30. The method of claim 29 , wherein the fungal cell is a yeast cell.

31. The method of claim 30 , wherein the yeast cell is a Saccharomyces sp. cell.

32. The method of claim 18 , wherein the genetically modified host cell is an algal, insect or mammalian cell line.

33. The method of claim 18 , wherein the IPP isomerase is encoded by the E. coli idi gene or the Saccharomyces cerevisiae idil gene; and the phosphatase is encoded by the E. coli nudB gene.

34. The method of claim 33 , wherein the genetically modified host cell is a prokaryotic cell.

35. The method of claim 34 , wherein the prokaryotic cell is selected from Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia , Vitreoscilla, Synechococcus, Synechocystis , or Paracoccus taxonomical classes.

36. The method of claim 34 , wherein the prokaryotic cell is an Escherichia coli cell.

Assignees

• Chou Howard
• Keasling Jay D
• Univ California

Inventors

• Chou Howard
• Keasling Jay D

Classifications

• C12N9/90

  Isomerases (5.) · CPC title

• C12P7/16 Primary

  Butanols · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12Y503/03002

  Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2) · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title