Identification of isoprene synthase variants with improved properties for the production of isoprene

• US 9,163,263 B2
• Filed: 03/13/2013
• Issued: 10/20/2015
• Est. Priority Date: 05/02/2012
• Status: Expired due to Fees

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company, Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Inventors: Beck, Zachary Q., Estell, David A., Miller, Jeffrey V., Rife, Christopher L., Wells, Derek H., Ngai, James
• Primary Examiner(s): NASHED, NASHAAT T
• Application Number:
  US13/801,971
• Publication Number:
  US 20130330796A1
• Time in Patent Office: 951 Days
• Field of Search: None
• US Class Current: 1/1
• 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 invention provides for compositions and methods for producing isoprene using isoprene synthase variants with improved properties.

Claims

1. A recombinant host cell comprising a nucleic acid encoding a polypeptide having isoprene synthase activity, wherein the polypeptide comprises one or more amino acid substitution(s) at one or more residue position(s) corresponding to positions in the amino acid sequence of SEQ ID NO:

1, wherein said one or more amino acid substitution(s) is selected from the group consisting of;

X118E, X36W, X22K, X228Y, X448L, X488F, X467H, X331P, X448I, X392Y, X448V, X282H, X383Y, X511 Y, X448E, X376M, X488L, X461A, X414I, X282W, X493E, X392S, X448Q, X282Y, X537N, X447Y, X240C, X538R, and X510C, and wherein the polypeptide has increased k_cat compared to a parent polypeptide which does not comprise said one or more amino acid substitution(s), and wherein said polypeptide has at least 80% sequence identity with SEQ ID NO;

1.

2. The host cell of claim 1 wherein the host cell is a bacterial, algal, or fungal 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 3 wherein the bacterial cell is selected from the group consisting of E. coli, L. acidophilus, P. citrea, B. subtilis, B. licheniformis, B. lentos, B. brevis, B. stearothennophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulars, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., P. alcaligenes, Clostridium sp., Corynebacterium sp., and C. glutamicum cell.

6. The host cell of claim 3, wherein the bacterial cell is a cyanobacterial or Clostridial cell.

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

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

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

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

11. The host cell of claim 9 wherein the fungal cell is a yeast cell.

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

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

14. A method for producing isoprene comprising (a) culturing the host cells of claim 1 under conditions suitable for the production of isoprene and (b) producing isoprene.

15. The method of claim 14 further comprising recovering the isoprene.