Isoprene synthase variants with improved solubility for production of isoprene

• US 8,735,134 B2
• Filed: 10/26/2012
• Issued: 05/27/2014
• Est. Priority Date: 10/27/2011
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Wells, Derek H., Rife, Christopher L.
• Primary Examiner(s): CHOWDHURY, IQBAL HOSSAIN
• Application Number:
  US13/662,328
• Publication Number:
  US 20130295632A1
• Time in Patent Office: 578 Days
• Field of Search: None
• US Class Current: 435/252.3
• CPC Class Codes: C08L 7/00 Compositions of natural rubber
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12Y 402/03007 Pentalenene synthase (4.2.3.7)

Abstract

The present invention provides methods and compositions of variant polypeptides having isoprene synthase activity with improved solubility. In particular, the present invention provides isoprene synthase variant for increased isoprene production in recombinant host cells.

Claims

1. A recombinant host cell comprising a polynucleotide encoding an isolated polypeptide variant having isoprene synthase activity, wherein the polypeptide variant comprises a substitution at residue X288 corresponding to SEQ ID NO:

1, and wherein the polypeptide has increased protein solubility compared to a parent polypeptide which does not comprise the substitution at residue X288, wherein said polypeptide variant is 90% identical to isoprene synthase polypeptide of SEQ ID NO;

1.

2. The host cell of claim 1 wherein the host cell is selected from the group consisting of a bacterial, algal, fungal, yeast, cyanobacterial, or Clostridial cell.

3. The host cell of claim 2 wherein the host cell is a bacterial cell.

4. The host cell of claim 3 wherein the bacterial cell is a gram-positive bacterial cell or gram-negative bacterial cell.

5. The host cell of claim 4 wherein the bacterial cell is selected from the group consisting of E. coli, L. acidophilus, 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., P. alcaligenes, C. glutamicum cells.

6. The host cell of claim 2 wherein the host cell is an algal cell.

7. The host cell of claim 6 wherein the algal cell is selected from the group consisting of green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, or dinoflagellates.

8. The host cell of claim 2 wherein the host cell is a fungal cell.

9. The host cell of claim 8 wherein the fungal cell is a filamentous fungi.

10. The host cell of claim 2 wherein the host cell is a yeast cell.

11. The host cell of claim 10 wherein the yeast cell is selected from the group consisting of Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.

12. The host cell of claim 11 wherein the yeast cell is a Saccharomyces cerevisiae cell.

13. An isolated variant polynucleotide that encodes the polypeptide variant of claim 1.

14. A vector comprising the polynucleotide of claim 13.

15. A recombinant host cell comprising the vector of claim 14.

16. The cell of claim 1 wherein the substitution is at S288.

17. The cell of claim 16 wherein the substitution is S288C.

18. The cell of claim 1, where the polypeptide has at least about 5% to at least about 75% increased protein solubility compared to the parent polypeptide.

19. The cell of claim 1 wherein the polypeptide is derived from a parent polypeptide isolated from a plant.

20. The cell of claim 19 wherein the parent polypeptide is isolated from a plant species selected from poplar (Populus sp.), kudzu (Pueraria sp.), English oak (Quercus sp.) or willow (Salix sp.).

21. The cell of claim 20 wherein the parent species is Populus sp.

22. The cell of claim 21 wherein in the parent is P. alba, P. tremuloides, P. trichocharpa, P. nigra.

23. The cell of claim 20 wherein the parent species is Pueraria sp.

24. The cell of claim 23 wherein in the parent species is Pueraria montana.

25. The cell of claim 20 wherein the parent species is Quercus sp.

26. The cell of claim 25 wherein in the parent species is Quercus rubur.

27. The cell of claim 20 wherein the parent species is Salix sp.

28. The cell of claim 27 wherein in the parent species is S. alba or S. baylonica.

29. A method of producing isoprene comprising:

(a) culturing the recombinant cell of claim 1 under conditions suitable for the production of isoprene and (b) producing isoprene.

30. A method of producing isoprene comprising:

(a) culturing the recombinant cell of claim 15 under conditions suitable for the production of isoprene and (b) producing isoprene.