Recombinant yeast host cells comprising FE-S cluster proteins

• US 9,206,447 B2
• Filed: 09/29/2009
• Issued: 12/08/2015
• Est. Priority Date: 09/29/2008
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax Advanced Biofuels LLC (International Flavors & Fragrances Incorporated)
• Inventors: Anthony, Larry Cameron, Tomb, Jean-Francois, Rothman, Steven Cary
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US12/569,069
• Publication Number:
  US 20100081179A1
• Time in Patent Office: 2,261 Days
• Field of Search: 435/135, 435/160, 435/254.11, 435/254.2, 435/254.21, 435/254.22, 435/254.23
• US Class Current: 1/1
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/26 Ketones
• 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

Yeast strains were engineered that have increased activity of heterologous proteins that require binding of an Fe—S cluster for their activity. The yeast strains have reduced activity of an endogenous Fe—S protein. Activities of heterologous fungal or plant 2Fe-2S dihydroxy-acid dehydratases and Fe—S propanediol dehydratase reactivase were increased for increased production of products made using biosynthetic pathways including these enzymes, such as valine, isoleucine, leucine, pantothenic acid (vitamin B5), isobutanol, 2-butanone and 2-butanol.

Claims

1. A recombinant yeast host cell comprising at least two engineered modifications comprising:

(i) a heterologous dihydroxy-acid dehydratase Fe—

S cluster protein expressed in the cytosol of the recombinant yeast host cell, wherein the heterologous dihydroxy-acid dehydratase Fe—

S cluster protein is a polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO;

114 using the Clustal W method of alimment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0,1, and Gonnet 250 series of protein weight matrix over the full length of the protein sequence and has a specific activity of at least 0.2 μ

mol min<sup>−

1</sup> mg<sup>−

1</sup>; and

(ii) an endogenous Fe—

S cluster protein with reduced expression, wherein the endogenous Fe—

S cluster protein is dihydroxy-acid dehydratase.

2. The recombinant yeast host cell of claim 1, wherein the yeast is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

3. The recombinant yeast host cell of claim 1, wherein the endogenous Fe—

S cluster protein is expressed in the mitochondria.

4. The recombinant yeast host cell of claim 1, wherein the host cell is Saccharomyces that expresses a gene encoding a polypeptide having the amino acid sequence as set forth in SEQ ID NO:

114.

5. The recombinant yeast host cell of claim 1, wherein the cell comprises an isobutanol biosynthetic pathway.

6. The recombinant yeast host cell of claim 5, wherein the cell produces isobutanol.

7. A method for the production of isobutanol comprising growing the recombinant yeast host cell of claim 6, under conditions wherein isobutanol is produced.

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

-ketoisovalerate comprising;

a) providing (1) the recombinant yeast host cell of claim 1; and

(2) a source of 2,3-dihydroxyisovalerate; and

b) growing the recombinant host cell of a) with said source of 2,3-dihydroxyisovalerate under conditions where the 2,3-dihydroxyisovalerate is converted by the host cell to α

-ketoisovalerate.