Increased Heterologous Fe-S Enzyme Activity in Yeast

US 20120064585A1
Filed: 11/15/2011
Published: 03/15/2012
Est. Priority Date: 09/29/2008
Status: Active Grant

First Claim

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1-24. -24. (canceled)

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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.

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42 Claims

1-24. -24. (canceled)

25. A recombinant yeast cell, comprising a heterologous Fe—
- S cluster dihydroxy-acid dehydratase (DHAD) comprising;
  
  (a) an amino acid sequence that matches a Profile Hidden Markov Model prepared using the amino acid sequences of SEQ ID NOs;
  
  175, 177, 179, 181, 183, 185, 187, and 189 with an E value of less than 10^−
  
  5; and
  
  (b) three conserved cysteine residues that correspond to positions 56, 129, and 201 of SEQ ID NO;
  
  179;
  
  wherein the heterologous Fe—
  
  S cluster DHAD is derived from Streptococcus mutans or Lactococcus lactis.
- View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
- - 26. The recombinant yeast cell of claim 25, wherein enzyme activity of DHAD is increased by at least 1.4 fold compared to a yeast cell which does not comprise the heterologous Fe—
    - S cluster DHAD.
  - 27. The recombinant yeast cell of claim 25, wherein the recombinant yeast cell further comprises a disruption in the ilv3 gene that encodes mitochondrial DHAD, resulting in reduced expression of endogenous mitochondrial DHAD.
  - 28. The recombinant yeast cell of claim 25, wherein the heterologous Fe—
    - S cluster DHAD comprises an amino acid sequence selected from the group consisting of;
      
      (i) an amino acid sequence having at least 90% identity to SEQ ID NO;
      
      179 or SEQ ID NO;
      
      187;
      
      (ii) an amino acid sequence encoded by a polynucleotide selected from the group consisting of;
      
      SEQ ID NO;
      
      178 or SEQ ID NO;
      
      186;
      
      a polynucleotide that is complementary to SEQ ID NO;
      
      178 or SEQ ID NO;
      
      186;
      
      a polynucleotide that hybridizes under stringent conditions to SEQ ID NO;
      
      178 or SEQ ID NO;
      
      186; and
      
      a polynucleotide having at least 90% identity to SEQ ID NO;
      
      178 or SEQ ID NO;
      
      186; and
      
      (iii) an amino acid sequence of (i) and (ii).
  - 29. The recombinant yeast cell of claim 25, wherein the recombinant yeast cell has a specific activity of DHAD of 0.2 to 0.35 μ
    - mol min^−
      
      1mg^−
      
      1.
  - 30. The recombinant yeast cell of claim 25, wherein the recombinant yeast cell further comprises an isobutanol biosynthetic pathway.
  - 31. The recombinant yeast cell of claim 30, wherein the isobutanol biosynthetic pathway comprises the following substrate to product conversions:
    - pyruvate to acetolactate;
      
      (ii) acetolactate to 2,3-dihydroxyisovalerate;
      
      (iii) 2,3-dihydroxyisovalerate to α
      
      -ketoisovalerate;
      
      (iv) α
      
      -ketoisovalerate to isobutyraldehyde; and
      
      (v) isobutyraldehyde to isobutanol.
  - 32. The recombinant yeast cell of claim 31, whereinthe substrate to product conversion of pyruvate to acetolactate is catalyzed by an acetolactate synthase;
    - the substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate is catalyzed by a ketol-acid reductoisomerase;
      
      the substrate to product conversion of 2,3-dihydroxyisovalerate to α
      
      -ketoisovalerate is catalyzed by the heterologous Fe—
      
      S cluster DHAD;
      
      the substrate to product conversion of α
      
      -ketoisovalerate to isobutyraldehyde is catalyzed by an α
      
      -keto acid decarboxylase; and
      
      the substrate to product conversion of isobutyraldehyde to isobutanol is catalyzed by an alcohol dehydrogenase.
  - 33. The recombinant yeast cell of claim 32, wherein at least two of said acetolactate synthase, said ketol-acid reductoisomerase, and said α
    - -keto acid decarboxylase are heterologous to the recombinant yeast cell.
  - 34. The recombinant yeast cell of claim 33, wherein at least two of said acetolactate synthase, said ketol-acid reductoisomerase, and said α
    - -keto acid decarboxylase enzymes are over-expressed in the recombinant yeast cell.
  - 35. The recombinant yeast cell of claim 25, wherein the yeast is Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, or Pichia.
  - 36. The recombinant yeast cell of claim 35, wherein the yeast is Saccharomyces cerevisiae.
  - 37. A method for the production of isobutanol, comprising:
    - (a) providing the recombinant yeast cell of claim 25; and
      
      (b) contacting the recombinant yeast cell of (a) under conditions wherein isobutanol is produced.
  - 38. The method of claim 37, further comprising:
    - (c) recovering the isobutanol.
  - 39. The method of claim 37, wherein the conditions wherein isobutanol is produced are anaerobic or microaerobic.
  - 40. A method for the conversion of 2,3-dihydroxyisovalerate to α
    - -ketoisovalerate, comprising;
      
      (a) providing the recombinant yeast cell of claim 25 and a source of 2,3-dihydroxyisovalerate; and
      
      (b) growing the recombinant yeast cell of (a) under conditions wherein the 2,3-dihydroxyisovalerate is converted to α
      
      -ketoisovalerate.
  - 41. The method of claim 40, wherein the recombinant yeast cell comprises a biosynthetic isobutanol pathway comprising the following substrate to product conversions:
    - (i) pyruvate to acetolactate;
      
      (ii) acetolactate to 2,3-dihydroxyisovalerate;
      
      (iii) 2,3-dihydroxyisovalerate to α
      
      -ketoisovalerate;
      
      (iv) α
      
      -ketoisovalerate to isobutyraldehyde; and
      
      (v) isobutyraldehyde to isobutanol;
      
      wherein the substrate to product conversion of 2,3-dihydroxyisovalerate to α
      
      -ketoisovalerate is catalyzed by the heterologous Fe—
      
      S cluster DHAD.
  - 42. The method of claim 41, wherein the heterologous Fe—
    - S cluster DHAD is derived from Streptococcus mutans or Lactococcus lactis.

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Current Assignee
Gevo Incorporated
Original Assignee
Butamax(TM) Advanced Biofuels LLC (BP PLC)
Inventors
Anthony, Larry Cameron, Maggio-Hall, Lori Ann, Rothman, Steven Cary, Tomb, Jean-Francois

Granted Patent

US 8,241,878 B2
Time in Patent Office

Days
Field of Search
US Class Current

435/137
CPC Class Codes

C12N 9/88   Lyases (4.)

C12P 7/16   Butanols

C12P 7/26   Ketones

C12P 7/40   containing a carboxyl group...

C12Y 402/01009   Dihydroxy-acid dehydratase ...

Y02E 50/10   Biofuels, e.g. bio-diesel

Increased Heterologous Fe-S Enzyme Activity in Yeast

First Claim

2 Assignments

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Abstract

8 Citations

42 Claims

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Increased Heterologous Fe-S Enzyme Activity in Yeast

First Claim

2 Assignments

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0 Petitions

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Accused Products

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Abstract

8 Citations

42 Claims

Specification

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