Three-dimensional structure of isoprene synthase and its use thereof for generating variants

• US 8,518,686 B2
• Filed: 04/22/2010
• Issued: 08/27/2013
• Est. Priority Date: 04/23/2009
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Beck, Zachary Quinn, Bott, Richard R., Rife, Christopher Lee, Wells, Derek H., Miller, Jeffrey V.
• Primary Examiner(s): Mondesi, Robert
• Assistant Examiner(s): CHOWDHURY, IQBAL HOSSAIN
• Application Number:
  US12/765,825
• Publication Number:
  US 20110076743A1
• Time in Patent Office: 1,223 Days
• Field of Search: None
• US Class Current: 435/252.33
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• 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 402/03027 Isoprene synthase (4.2.3.27)

Abstract

The present invention provides a three-dimensional structures of P. tremuloides isoprene synthase and P. alba isoprene synthase. The invention also provides methods of using the three-dimensional structure to design isoprene synthases with improved activity for increased isoprene production in microbial host cells. Biosynthetically produced isoprene of the present invention finds use in the manufacture of rubber and elastomers.

Claims

1. An isolated host cell comprising an isoprene synthase variant produced by:

a) identifying hydrophobic amino acid residues on the surface of isoprene synthase based on the three dimensional structure of P. alba isoprene synthase;

b) introducing one or more amino acid substitutions in the surface amino acids of said isoprene synthase to produce an isoprene synthase variant; and

c) identifying said isoprene synthase variants with substitutions that improve activity, expression or stability of the isoprene synthase,

wherein said isoprene synthase variant comprises one or more amino acid substitutions at a hydrophobic surface amino acid position corresponding to a hydrophobic surface amino acid position of SEQ ID NO;

45 selected from the group I28, V30, L130, G153, L469, L303 and L494; and

wherein said isoprene synthase variant has improved activity, expression or stability of the isoprene synthase compared to an isoprene synthase without said one or more amino acid substitutions.

15. A method of producing isoprene, comprising:

(a) providing a host cell of any one of claims 1, 2 and 3-14; and

(b) culturing the host cell under conditions suitable for producing isoprene.

2. An isolated host cell comprising an isoprene synthase variant, wherein the isoprene synthase variant comprises one or more amino acid substitutions at a hydrophobic surface amino acid position corresponding to a hydrophobic surface amino acid position of SEQ ID NO:

45 selected from the group I28, V30, L130, G153, L469, L303 and L494 and wherein said isoprene synthase variant has improved activity, expression and/or stability as compared to an isoprene synthase without said one or more amino acid substitutions.

3. The isolated host cell of claim 2 wherein the variant comprises one or more substitutions selected from the group consisting of:

I28W, I28T, I28R, I28Y, V30K, L130W, L130K, L130S, L130Y, L130R, L130V, L130I, L130E, L130D, G153K, G153H, G153L, G153W, L303I, L469A, L469Q, L494P, L494C, L494I, L494V, L494S, L494G, and L494D.

4. The isolated host cell of claim 2, wherein the cell is a gram-positive bacterial cell, gram-negative bacterial cell, filamentous fungal cell, or yeast cell.

5. The isolated host cell of claim 4, wherein the bacterial cell is 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.

6. The host cell of claim 4 wherein the cell is selected from the group consisting of Escherichia sp. (E. coli), Saccharomyces sp. (S. cerevisiae), Panteoa sp. (P. citrea), Bacillus sp. (B. subtilis), Yarrowia sp. (Y. lipolytica), and Trichoderma (T. reesei).

7. The isolated host cell of claim 2, wherein the host cell further comprises a heterologous or native nucleic acid encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide and/or a heterologous or native nucleic acid encoding a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide, optionally in combination with the native 1-deoxy-D-xylulose-5-phosphate (DXP) pathway.

8. The isolated host cell of claim 2, wherein the host cell further comprises one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide.

9. The isolated host cell of claim 2, wherein the host cell comprises one vector encoding the isoprene synthase variant, the IDI polypeptide, and the DXS polypeptide.

10. The isolated host cell of claim 9 wherein the host cell further comprises a heterologous nucleic acid encoding a mevalonate (MVA) pathway polypeptide selected from the group consisting of an MVA pathway polypeptide from Saccharomyces cerevisiae and Enterococcus faecalis.

11. The isolated host cell of claim 2 wherein the host cell further comprises one or more nucleic acids encoding an MVA pathway polypeptide and a DXS polypeptide and wherein one vector encodes the isoprene synthase variant, the MVA pathway polypeptide, and the DXS polypeptide.

12. The isolated host cell of claim 2 wherein the host cell further comprises one or more nucleic acids encoding a DXS polypeptide, an IDI polypeptide, or one or more of the rest of the DXP pathway polypeptides, and a MVA pathway polypeptide.

13. The isolated host cell of claim 2, wherein the host cell further comprises one or more heterologous nucleic acid(s) encoding an MVA pathway polypeptide.

14. The isolated host cell of claim 13, wherein the host cell further comprises one or more nucleic acids encoding an IDI polypeptide.