Activity of Fe—S cluster requiring proteins

• US 9,297,016 B2
• Filed: 02/17/2011
• Issued: 03/29/2016
• Est. Priority Date: 02/17/2010
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Flint, Dennis, Paul, Brian James, Ye, Rick W.
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US13/029,558
• Publication Number:
  US 20120064561A1
• Time in Patent Office: 1,867 Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 13/02 Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes peptides C12P21/00 or C07K
• CPC Class Codes: C12P 13/04 Alpha- or beta- amino acids other amino acids C12P13/005
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12Q 1/527 involving lyase
• CPC Class Codes: C12Y 402/01009 Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention is related to a recombinant host cell, in particular a yeast cell, comprising a dihydroxy-acid dehydratase polypeptide. The invention is also related to a recombinant host cell having increased specific activity of the dihydroxy-acid dehydratase polypeptide as a result of increased expression of the polypeptide, modulation of the Fe—S cluster biosynthesis of the cell, or a combination thereof. The present invention also includes methods of using the host cells, as well as, methods for identifying polypeptides that increase the flux in an Fe—S cluster biosynthesis pathway in a host cell.

Claims

1. A recombinant yeast host cell comprising:

(a) at least one heterologous polynucleotide encoding a dihydroxy-acid dehydratase enzyme (DHAD), wherein the dihydroxy-acid dehydratase enzyme has an amino acid sequence with at least 95% identity to SEQ ID NO;

168; and

(b) at least one deletion in an endogenous gene encoding a polypeptide affecting Fe—

S cluster biosynthesis, wherein the gene encoding the polypeptide affecting Fe—

S cluster biosynthesis is FRA2.

2. The recombinant yeast host cell of claim 1, wherein said at least one heterologous polynucleotide encoding a dihydroxy-acid dehydratase enzyme is integrated at least once in the recombinant yeast host cell DNA.

3. The recombinant yeast host cell of claim 1, wherein the dihydroxy-acid dehydratase enzyme is expressed in the cytosol of the recombinant yeast host cell.

4. The recombinant yeast host cell of claim 1, wherein the dihydroxy-acid dehydratase enzyme has the amino acid sequence of SEQ ID NO:

168.

5. The recombinant yeast host cell of claim 1, wherein said recombinant yeast host cell produces isobutanol.

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

7. The recombinant yeast host cell of claim 1, wherein monomers of the dihydroxy-acid dehydratase enzyme have an Fe—

S cluster loading selected from the group consisting of;

(a) at least 10%;

(b) at least 15%;

(c) at least 20%;

(d) at least 25%;

(e) at least 30%;

(f) at least 35%;

(g) at least 40%;

(h) at least 45%;

(i) at least 50%;

(j) at least 60%;

(k) at least 70%;

(l) at least 80%;

(m) at least 90%; and

(n) at least 95%.

8. A method of making a product comprising:

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

(b) contacting the recombinant yeast host cell of (a) with a fermentable carbon substrate in a fermentation medium under conditions wherein said product is produced;

wherein the product is selected from the group consisting of branched chain amino acids, pantothenic acid, 2-methyl-1-butanol, 3-methyl-l-butanol, isobutanol, and combinations thereof.

9. A method of making isobutanol comprising:

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

(b) contacting the recombinant yeast host cell of (a) with a fermentable carbon substrate in a fermentation medium under conditions wherein isobutanol is produced.

10. A method for the conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate comprising:

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

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

wherein 2,3-dihydroxyisovalerate is converted to a-ketoisovalerate.

11. The method of claim 10, wherein said conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate compared to a control yeast host cell containing at least one heterologous polynucleotide encoding dihydroxy-acid dehydratase is increased in an amount selected from the group consisting of:

(a) at least 5%;

(b) at least 10%;

(c) at least 15%;

(d) at least 20%;

(e) at least 25%;

(f) at least 30%;

(g) at least 35%;

(h) at least 40%;

(i) at least 45%;

(j) at least 50%;

(k) at least 60%;

(l) at least 70%;

(m) at least 80%;

(n) at least 90%; and

(o) at least 95%.

12. A method of increasing the catalytic activity of DHAD in a recombinant yeast host cell comprising:

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

(b) growing the recombinant yeast host cell of (a) under conditions whereby the catalytic activity of the DHAD is increased.

13. The method of claim 12, wherein said increased catalytic activity is selected from the group consisting of:

(a) greater than 10%;

(b) greater than 20%;

(c) greater than 30%;

(d) greater than 40%;

(e) greater than 50%;

(f) greater than 60%;

(g) greater than 70%;

(h) greater than 80%;

(i) greater than 90%; and

(j) greater than 95%.