Ketol-acid reductoisomerase enzymes and methods of use

• US 9,388,392 B2
• Filed: 09/14/2015
• Issued: 07/12/2016
• Est. Priority Date: 05/11/2012
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Govindarajan, Sridhar, Li, Yougen, Liao, Der-Ing, O'Keefe, Daniel P., Minshull, Jeremy Stephen, Rothman, Steven Cary, Tobias, Alexander Vincent
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US14/853,108
• Publication Number:
  US 20160032255A1
• Time in Patent Office: 302 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 13/02 Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes peptides C12P21/00 or C07K
• CPC Class Codes: C12P 13/06 Alanine; Leucine; Isoleucine; Serine; Homoserine
• CPC Class Codes: C12P 13/08 Lysine; Diaminopimelic acid; Threonine; Valine
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12P 7/44 Polycarboxylic acids
• CPC Class Codes: C12Y 101/01086 Ketol-acid reductoisomerase (1.1.1.86)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Provided herein are polypeptides having ketol-acid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

Claims

1. A polypeptide having ketol-acid reductoisomerase activity, wherein the polypeptide comprises

a. the amino acid sequence of SEQ ID NO:

42;

b. at least 99% identity to SEQ ID NO;

42;

c. at least 99% identity to SEQ ID NO;

42 and at least one of the following substitutions;

M301I, Y239H, Y113F, S322A, A71K, N114G, A329R, A69T, N114S, G72W, A295V, E264K, R280D, A329E, S157T, M238I, Q272T, K335M, R280H, and a combination thereof;

or

d. an active fragment of any one of (a) to (c).

2. The polypeptide of claim 1 further comprising a substitution at at least one of position L33, P47, F50, F61, I80, and V156.

3. The polypeptide of claim 1 further comprising at least one of the following substitutions:

I13L, P47Y, F50A, V53A, S86A, A268E, V76I, L88V, and a combination thereof.

4. A recombinant microbial host cell comprising the polypeptide of claim 1.

5. The recombinant microbial host cell of claim 4, wherein the recombinant microbial host cell further comprises reduced or eliminated acetolactate reductase activity.

6. The recombinant microbial host cell of claim 5, wherein the recombinant microbial host cell further comprises at least one deletion, mutation, and/or substitution in fra2.

7. The recombinant microbial host cell of claim 4 further comprising the substrate to product conversions:

a. pyruvate to acetolactate

b. 2,3-dihydroxyisovalerate to α

-ketoisovalerate

c. α

-ketoisovalerate to isobutyraldehyde; and

d. isobutyraldehyde to isobutanol.

8. The recombinant microbial host cell of claim 4, wherein the recombinant microbial host cell is a yeast host cell.

9. The recombinant microbial host cell of claim 8, wherein the recombinant microbial host cell is Saccharomyces cerevisiae.

10. A method for converting acetolactate to dihydroxyisovalerate comprising:

a. providing a microbial host cell comprising a polypeptide of claim 1;

b. contacting the polypeptide with acetolactate wherein dihydroxyisovalerate is produced.

11. A method of producing a product selected from the group consisting of isobutanol, pantothenate, valine, leucine, isoleucine, 3,3-dimethylmalate, 2-methyl-1-butanol or combinations thereof comprising:

a. providing the recombinant microbial host cell of claim 4, wherein the recombinant host cell comprises a product biosynthetic pathway; and

b. contacting the recombinant microbial host cell with a carbon substrate under conditions whereby the product is produced.

12. The method of claim 11, wherein at least a portion of the contacting occurs under anaerobic conditions.