DHAD variants for butanol production

• US 9,650,624 B2
• Filed: 12/27/2013
• Issued: 05/16/2017
• Est. Priority Date: 12/28/2012
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Maggio-Hall, Lori Ann, Paul, Brian James, Ye, Rick W., Rothman, Steven Cary
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US14/142,398
• Publication Number:
  US 20140186910A1
• Time in Patent Office: 1,236 Days
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12Y 402/01009 Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol.

Claims

1. An isolated polypeptide or fragment thereof having dihydroxy-acid dehydratase (DHAD) activity, wherein the polypeptide or fragment thereof comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO:

565, and wherein the amino acid sequence comprises a deletion of about 5 to about 20 amino acids from the C-terminal end of the amino acid sequence.

2. The isolated polypeptide or fragment thereof of claim 1, wherein the amino acid sequence further comprises one or more amino acid substitutions selected from P378A, P378G, P378V, P378I, P378L, G383S, G383A, G383V, G383L, G383I, I387V, I387M, I387L, I387G, I387A, L388I, L388V, L388A, and L388M.

3. The isolated polypeptide or fragment thereof of claim 1, wherein the polypeptide or fragment thereof having DHAD activity catalyzes the conversion of 2,3-dihydroxyisovalerate to α

-ketoisovalerate.

4. The isolated polypeptide or fragment thereof of claim 1, further comprising a polycysteine or polyhistidine tag.

5. The isolated polypeptide or fragment thereof of claim 4, further comprising the amino acid sequence of SEQ ID NO:

589.

6. The isolated polypeptide or fragment thereof of claim 2, wherein the amino acid motif comprises the amino acid sequence of SEQ ID NO:

573.

7. The isolated polypeptide or fragment thereof of claim 1, wherein the polypeptide or fragment thereof having DHAD activity is a [2Fe-2S]²⁺DHAD.

8. The isolated polypeptide or fragment thereof of claim 1, wherein the polypeptide or fragment thereof having DHAD activity has an amino acid sequence that matches the Profile Hidden Markov Model (HMM) of Table 6 with an E value of <

10⁵.

9. The isolated polypeptide or fragment thereof of claim 1, wherein the polypeptide or fragment thereof having DHAD activity comprises three conserved cysteines corresponding to positions 56, 129, and 201 in the amino acid sequences of the Streptococcus mutans DHAD corresponding to SEQ ID NO:

544.

10. The isolated polypeptide or fragment thereof of claim 1, wherein the polypeptide or fragment further comprises a polypeptide sequence selected from SEQ ID NO:

723, SEQ ID NO;

724, SEQ ID NO;

725, SEQ ID NO;

726, SEQ ID NO;

727, SEQ ID NO;

728, SEQ ID NO;

729, SEQ ID NO;

730, SEQ ID NO;

731, SEQ ID NO;

732, SEQ ID NO;

733, SEQ ID NO;

734, SEQ ID NO;

735, SEQ ID NO;

736, SEQ ID NO;

737, SEQ ID NO;

738, SEQ ID NO;

739, SEQ ID NO;

740, SEQ ID NO;

741, SEQ ID NO;

742, SEQ ID NO;

743, SEQ ID NO;

744, SEQ ID NO;

745, SEQ ID NO;

746, SEQ ID NO;

747, and SEQ ID NO;

748.

11. The isolated polypeptide or fragment thereof of claim 1, wherein the amino acid sequence comprises a deletion of about 5 to about 15 amino acids from the C-terminal end of the amino acid sequence.

12. The isolated polypeptide or fragment thereof of claim 1, wherein the amino acid sequence comprises a deletion of 9 C-terminal amino acids.