Biological production of pentose sugars using recombinant cells

• US 8,691,541 B2
• Filed: 12/22/2011
• Issued: 04/08/2014
• Est. Priority Date: 12/22/2010
• Status: Expired due to Fees

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: McAuliffe, Joseph C., Muir, Rachel E.
• Primary Examiner(s): Saidha, Tekchand
• Assistant Examiner(s): Ramanujam, Rama P
• Application Number:
  US13/335,792
• Publication Number:
  US 20120329102A1
• Time in Patent Office: 838 Days
• Field of Search: 435/167, 435/243
• US Class Current: 435/167
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12N 1/18 Baker's yeast; Brewer's yeast
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/24 produced by the action of an isomerase, e.g. fructose

Abstract

The invention provides, inter alia, compositions and methods for the biological production of pentose sugars, such as 2-methylerythritol (2-ME), 1-deoxyxylulose (1-DX), and monoacetylated-2-C-methylerythritols, using recombinant cells.

Claims

1. A method of producing a pentose sugar, the method comprising:

(a) culturing recombinant cells comprising (i) a heterologous nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide and/or (ii) more than one copy of an endogenous nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide, wherein the cells are cultured under conditions suitable for producing a pentose sugar;

or

(b) culturing recombinant cells comprising (i) a heterologous nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide and (ii) one or more copies of an endogenous nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide, wherein the cells are cultured under conditions suitable for producing a pentose sugar; and

(c) producing said pentose sugar.

2. The method of claim 1 wherein the recombinant cells further comprise (iii) a heterologous nucleic acid encoding a phosphatase and/or (iv) one or more copies of an endogenous nucleic acid encoding a phosphatase.

3. The method of claim 2, wherein the pentose sugar is selected from the group consisting of 2-methylerythritol (2-ME) and 1-deoxyxylulose (1-DX).

4. The method of claim 3, wherein the pentose sugar is 2-methylerythritol (2-ME).

5. The method of claim 4, wherein the recombinant cells are capable of producing a cumulative titer of 2-ME of at least about 20 g/L.

6. The method of claim 4, wherein the recombinant cells are capable of producing a cumulative titer of 2-ME of at least about 30 g/L.

7. The method of claim 4, wherein the recombinant cells are capable of producing a cumulative titer of 2-ME of at least about 45 g/L.

8. The method of claim 1, wherein the recombinant cells further comprise (iii) one or more heterologous nucleic acids encoding a deoxyxylulose phosphate pathway polypeptide other than a 1-deoxyxylulose-5-phosphate synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide and/or (iv) one or more copies of an endogenous nucleic acid encoding a deoxyxylulose phosphate pathway polypeptide other than a 1-deoxyxylulose-5-phosphate synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide.

9. The method of claim 8, wherein the deoxyxylulose phosphate pathway polypeptide is 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase.

10. The method of claim 9, wherein the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase polypeptide is a T. elongatus 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase polypeptide.

11. The method of claim 9, wherein the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase polypeptide is an E. coli 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase polypeptide.

12. The method of claim 8, wherein the recombinant cells further comprise a nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide.

13. The method of claim 12, wherein the iron-sulfur cluster-interacting redox polypeptide is selected from ferrodoxin and flavodoxin.

14. The method of claim 1, wherein the recombinant cells further comprise at least one heterologous nucleic acid encoding an isopentenyl-diphosphate delta-isomerase polypeptide or at least one copy of an endogenous nucleic acid encoding an isopentenyl-diphosphate delta-isomerase polypeptide.

15. The method of claim 1, wherein the recombinant cells are bacterial, algal, fungal or yeast cells.

16. The method of claim 15, wherein the cells are bacterial cells.

17. The method of claim 16, wherein the bacterial cells are gram-positive bacterial cells or gram-negative bacterial cells.

18. The method claim 17, wherein the bacterial cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

19. The method of claim 18, wherein the bacterial cells is an E. coli cells.

20. The method of claim 8, wherein the deoxyxylulose phosphate pathway polypeptide is selected from the group of diphosphocytidyl-2C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, and isopentenyl-diphosphate delta-isomerase.

21. The method of claim 20, wherein the additional deoxyxylulose phosphate pathway polypeptide is selected from the group of 1 hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, and isopentenyl-diphosphate delta-isomerase.

22. A cell culture comprising the recombinant cells of claim 1, wherein the cell culture produces at least about 45 g/L of 2-ME.