Identification and use of bacterial [2Fe-2S] dihydroxy-acid dehydratases

• US 8,951,937 B2
• Filed: 03/15/2013
• Issued: 02/10/2015
• Est. Priority Date: 09/29/2008
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Flint, Dennis, Rothman, Steven Cary, Suh, Wonchul, Tomb, Jean-Francois, Ye, Rick W.
• Primary Examiner(s): RAMIREZ, DELIA M
• Application Number:
  US13/838,508
• Publication Number:
  US 20140030776A1
• Time in Patent Office: 697 Days
• Field of Search: 435/232, 435/69.1, 435/320.1, 435/325, 435/252.3
• US Class Current: 506/7
• CPC Class Codes: C12N 9/88 Lyases (4.)
• 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 7/40 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: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

A group of bacterial dihydroxy-acid dehydratases having a [2Fe-2S] cluster was discovered. Bacterial [2Fe-2S] DHADs were expressed as heterologous proteins in bacteria and yeast cells, providing DHAD activity for conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate. Isobutanol and other compounds may be synthesized in pathways that include bacterial [2Fe-2S] DHAD activity.

Claims

1. A method for identifying [2Fe-2S] dihydroxy-acid dehydratase (DHAD) enzymes comprising:

(a) querying one or more amino acid sequences with a Profile Hidden Markov Model prepared using the proteins of SEQ ID NO;

164, 168, 230, 232, 298, 310, 344, and 346, wherein amino acid sequences with an E-value of less than 10<sup>−

5</sup> provide a first subset of sequences;

(b) analyzing the first subset of sequences of step (a) for the presence of three conserved cysteines that correspond to positions 56, 129, and 201 in SEQ ID NO;

168, whereby a second subset of sequences is identified;

(c) analyzing the second subset of sequences of step (b) for the presence of signature conserved amino acids at positions corresponding to positions in SEQ ID NO;

168, wherein said amino acids are aspartic acid at position 88, arginine or asparagine at position 142, asparagine at position 208, and leucine at position 454, whereby a third subset of sequences is identified;

(d) expressing a protein having an amino acid sequence identified by step (b) or step (c) in a host cell;

(e) purifying the protein of step (d);

(f) confirming that the protein of step (e) has DHAD activity; and

(g) confirming that the protein of step (f) is a [2Fe-2S] DHAD enzyme by UV-vis and EPR spectroscopy.

2. The method of claim 1, wherein the [2Fe-2S] DHAD enzyme is a bacterial [2Fe-2S] DHAD enzyme.

3. The method of claim 1, wherein the host cell lacks endogenous DHAD activity.

4. The method of claim 2, wherein the host cell is a bacterial cell or a yeast cell.

5. The method of claim 4, wherein the bacterial cell is a member of a genus of bacteria selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

6. The method of claim 4, wherein the yeast cell is a member of a genus of yeast selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, and Pichia.

7. The method of claim 4, wherein the host cell further comprises an isobutanol biosynthetic pathway.