Ketol-acid reductoisomerase enzymes and methods of use
View Patent ↗Provided herein are polypeptides having ketol-aid 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.
1. A polypeptide having ketol-acid reductoisomerase activity, wherein the polypeptide comprises
a. the amino acid sequence of SEQ ID NO: 41;
b. at least 99% identity to SEQ ID NO: 41;
c. at least 99% identity to SEQ ID NO: 41 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 said host cell further comprises reduced or eliminated acetolactate reductase activity.
6. The recombinant microbial host cell of claim 5 wherein said 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 said host cell is a yeast host cell.
9. The recombinant microbial host cell of claim 8 wherein said 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, and 2-methyl- 1-butanol or combinations thereof comprising:
a. providing the recombinant host cell of claim 4 wherein the recombinant host cell comprises a product biosynthetic pathway; and
b. contacting the 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.