IDENTIFICATION AND USE OF BACTERIAL [2Fe-2S] DIHYDROXY-ACID DEHYDRATASES

• US 20100081154A1
• Filed: 09/29/2009
• Published: 04/01/2010
• Est. Priority Date: 09/29/2008
• Status: Abandoned Application

• Current Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: FLINT, DENNIS, Ye, Rick W., Suh, Wonchul, Tomb, Jean-Francois, Rothman, Steven Cary
• Application Number:
  US12/569,636
• Publication Number:
  US 20100081154A1
• Time in Patent Office: Days
• US Class Current: 435/18
• 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] DHAD enzymes comprising:

a) querying one or more amino acid sequences with a Profile Hidden Markov Model prepared using the proteins of SEQ ID NOs;

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

5</sup> provides a first subset of sequences whereby said first subset of sequences correspond to one or more DHAD related proteins;

b) analyzing the first subset of sequences that correspond to one or more DHAD related proteins of step (a) for the presence of three conserved cysteines that correspond to positions 56, 129, and 201 in the Streptococcus mutans dihydroxy-acid dehydratase amino acid sequence (SEQ ID NO;

     168) whereby a second subset of sequences encoding [2Fe-2S] DHAD enzymes are identified; and

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

     168) that 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 encoding [2Fe-2S] DHAD enzymes are further identified.

2. The method of claim 1 further comprising

d) expressing a polypeptide having a sequence identifiable by any one or all of steps a), b), and c) in a cell;

and

e) confirming that said polypeptide has DHAD activity in the cell.

3. The method of claim 1 further comprising

d) purifying a protein encoded by a sequence identifiable by any one or all of steps a), b), and c);

and

e) confirming that said protein is a [2Fe-2S] DHAD enzyme by UV-vis and EPR spectroscopy.

4. The method of claim 1 further comprising selecting one or more sequences corresponding to bacterial [2Fe-2S] DHAD enzyme sequences identified in any one or all of steps a), b), and c).

5. The method of claim 2 wherein the cell lacks endogenous DHAD activity.

6. The method of claim 4 further comprising

d) expressing said selected one or more sequences corresponding to bacterial [2Fe-2S] DHAD enzyme sequences in a cell;

and

e) confirming that said enzyme sequence has DHAD activity in the cell.

7. The method of claim 4 further comprising

d) purifying a protein encoded by said selected one or more sequences corresponding to bacterial [2Fe-2S] DHAD enzyme sequences whereby a purified protein is produced;

and

e) confirming that the protein is a [2Fe-2S] DHAD enzyme by UV-vis and EPR spectroscopy.

8. A microbial host cell comprising at least one heterologous [2Fe-2S] DHAD enzyme identifiable by the method of any one of claims 1-7.

9. The microbial host cell of claim 8 wherein the cell is bacterial cell or a yeast cell.

10. The microbial host cell of claim 9 wherein the bacterial host 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, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

11. The microbial host cell of claim 9 wherein the yeast cells a member of a genus of yeast selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

12. The microbial host cell of claim 8 wherein the cell produces isobutanol.

13. A method for the production of isobutanol comprising:

a) providing the microbial host cell of claim 8 wherein said host cell comprises an isobutanol biosynthetic pathway; and

b) growing the host cell of step (a) under conditions wherein isobutanol is produced.

14. A method for the conversion of 2,3-dihydroxyisovalerate to α

-ketoisovalerate comprising;

a) providing the microbial host of claim 8 and a source of 2,3-dihydroxyisovalerate; and

b) growing the microbial host cell of (a) under conditions where the 2,3-dihydroxyisovalerate is converted to α

-ketoisovalerate.