Ketol-acid reductoisomerase enzymes and methods of use

Patent metadata

Field	Value
Publication number	US-9388392-B2
Application number	US-201514853108-A
Country	US
Kind code	B2
Filing date	Sep 14, 2015
Priority date	May 11, 2012
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

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.

First claim

What is claimed is:

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.

Assignees

• Butamax Advanced Biofuels Llc

Inventors

• Govindarajan Sridhar
• Li Yougen
• Liao Der-Ing
• O'Keefe Daniel P
• Minshull Jeremy Stephen
• Rothman Steven Cary
• Tobias Alexander Vincent

Classifications

• C12P13/02

  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 title

• C12P13/08

  Lysine; Diaminopimelic acid; Threonine; Valine · CPC title

• C12Y101/01086

  Ketol-acid reductoisomerase (1.1.1.86) · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title