Polypeptides with ketol-acid reductoisomerase activity

• US 10,174,345 B2
• Filed: 10/27/2016
• Issued: 01/08/2019
• Est. Priority Date: 09/26/2012
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax Advanced Biofuels LLC (International Flavors & Fragrances Incorporated)
• Inventors: Li, Yougen, McElvain, Jessica, Rothman, Steven Cary
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US15/335,628
• Publication Number:
  US 20170044578A1
• Time in Patent Office: 803 Days
• Field of Search: 435160, 43525421, 4353201
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12Y 101/01001 Alcohol dehydrogenase (1.1.1.1)
• CPC Class Codes: C12Y 101/01086 Ketol-acid reductoisomerase (1.1.1.86)
• CPC Class Codes: C12Y 102/01003 Aldehyde dehydrogenase (NAD+) (1.2.1.3)
• CPC Class Codes: C12Y 401/01001 Pyruvate decarboxylase (4.1.1.1)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Polypeptides having ketol-acid reductoisomerase activity are provided. Also disclosed are recombinant host cells comprising isobutanol biosynthetic pathways employing such polypeptides. Methods for producing isobutanol employing host cells comprising the polypeptides having ketol-acid reductoisomerase activity are also disclosed.

Claims

1. A recombinant yeast host cell comprising an isobutanol biosynthetic pathway and a heterologous polypeptide with ketol-acid reductoisomerase activity having at least 99% identity to SEQ ID NO:

553 or a heterologous polynucleotide encoding said heterologous polypeptide.

2. The recombinant yeast host cell of claim 1, wherein the yeast is selected from the group consisting of yeast cell is a member of a genus of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia, or Pichia.

3. The recombinant yeast host cell of claim 1, wherein the isobutanol production pathway comprises the following substrate to product conversions:

a. pyruvate to acetolactate

b. acetolactate to 2,3-dihydroxyisovalerate

c. 2,3-dihydroxyisovalerate to 2-ketoisovalerate

d. 2-ketoisovalerate to isobutyraldehyde; and

e. isobutyraldehyde to isobutanol

wherein more than one of the substrate to product conversions is catalyzed by an enzyme that is heterologous to the host cell.

4. The recombinant yeast host cell of claim 3 wherein all of the substrate to product conversions are catalyzed by enzymes heterologous to the host cell.

5. The recombinant yeast host cell of claim 3 wherein the substrate to product conversion for isobutyraldehyde to isobutanol is catalyzed by an alcohol dehydrogenase enzyme which utilizes NADH as a cofactor.

6. The recombinant yeast host cell of claim 1, wherein the host cell has reduced or eliminated acetolactate reductase activity.

7. The recombinant yeast host cell of claim 1, wherein the host cell has reduced or eliminated aldehyde dehydrogenase activity.

8. The recombinant yeast host cell of claim 1, wherein the host cell is yeast and has reduced or eliminated pyruvate decarboxylase activity.

9. The recombinant yeast host cell of claim 1 comprising an isobutanol production pathway comprising the following substrate to product conversions:

a. pyruvate to acetolactate

b. acetolactate to 2,3-dihydroxyisovalerate

c. 2,3-dihydroxyisovalerate to 2-ketoisovalerate

d. 2-ketoisovalerate to isobutyraldehyde; and

e. isobutyraldehyde to isobutanol

wherein the substrate to product conversions are catalyzed by enzymes substantially localized to the cytosol.

10. A composition comprising isobutanol and a recombinant yeast host cell of claim 1.

11. The recombinant yeast host cell of claim 1, wherein the polypeptide comprises at least one substitution at at least one of the following positions:

S32, H37, K42, G45, G66, T71, A73, A79, A92, M94, D98, E100, A105, E147, E148, V156, G164, L167, M169, A176, R181, V184, T187, T191, A214, V220, L243, Y254, N258, E274, K278, A279, G304, or M312.

12. The recombinant yeast host cell of claim 11, wherein the polypeptide comprises at least one of the following substitutions:

S32Y, H37N, K42N, G45C, G66A, T71S, A73T, A79D, A92D, M941, M94L, D98V, E100Q, A105T, E147V, E148G, E148Q, V156A, G164D, L167M, M169F, M169K, M169L, A176T, R181K, V184I, T187S, T191N, T191S, A214V, V220I, L243S, Y254F, N258S, E274, K278M, A279T, G304V, or M312K.