Recombinant yeast host cell with Fe-S cluster proteins and methods of using thereof
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First Claim
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1. A method of converting 2,3-dihydroxyisovalerate to α
- -ketoisovalerate, comprising providing a recombinant yeast host cell expressing a heterologous dihydroxy-acid dehydratase (DHAD) protein that comprises(i) an amino acid sequence having at least 95% identity to SEQ ID NO;
179 or 187; and
(ii) three conserved cysteine residues that correspond to positions 56, 129, and 201 of SEQ ID NO;
179;
wherein the substrate 2,3-dihydroxyisovalerate is present in the yeast host cell and wherein the expressed heterologous DHAD protein catalyzes the conversion of 2,3-dihydroxyisovalerate to α
-ketoisovalerate.
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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.
60 Citations
15 Claims
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1. A method of converting 2,3-dihydroxyisovalerate to α
- -ketoisovalerate, comprising providing a recombinant yeast host cell expressing a heterologous dihydroxy-acid dehydratase (DHAD) protein that comprises
(i) an amino acid sequence having at least 95% identity to SEQ ID NO;
179 or 187; and(ii) three conserved cysteine residues that correspond to positions 56, 129, and 201 of SEQ ID NO;
179;wherein the substrate 2,3-dihydroxyisovalerate is present in the yeast host cell and wherein the expressed heterologous DHAD protein catalyzes the conversion of 2,3-dihydroxyisovalerate to α
-ketoisovalerate.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
and wherein (a) the substrate to product conversion of step (i) is catalyzed by an acetolactate synthase; (b) the substrate to product conversion of step (ii) is catalyzed by a ketol-acid reductoisomerase; (c) the substrate to product conversion of step (iii) is catalyzed by the heterologous DHAD; (d) the substrate to product conversion of step (iv) is catalyzed by an α
-keto acid decarboxylase; and(e) the substrate to product conversion of step (v) is catalyzed by an alcohol dehydrogenase.
- -ketoisovalerate, comprising providing a recombinant yeast host cell expressing a heterologous dihydroxy-acid dehydratase (DHAD) protein that comprises
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7. The method of claim 6, wherein two or more of the enzymes (a), (b), and (d) are heterologous to the recombinant yeast host cell.
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8. The method of claim 7, wherein two or more of the enzymes (a), (b), and (d) are over-expressed in the recombinant yeast host cell.
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9. The method of claim 1, wherein the yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, and Pichia.
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10. The method of claim 9, wherein the yeast host cell is Saccharomyces cerevisiae.
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11. The method of claim 6, further comprising:
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(a) culturing the recombinant yeast host cell under suitable conditions to produce isobutanol from pyruvate; and (b) recovering the isobutanol.
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12. The method of claim 11, wherein said recovering is by distillation, liquid-liquid extraction, adsorption, decantation, pervaporation or combinations thereof.
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13. The method of claim 11, further comprising (c) removing solids from the fermentation medium.
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14. The method of claim 13, wherein said removing is by centrifugation, filtration or decantation.
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15. The method of claim 13, wherein said removing step (c) occurs before said recovering step (b).
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Litigation Data
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Current AssigneeGevo Incorporated
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Original AssigneeButamax(TM) Advanced Biofuels LLC (BP PLC)
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InventorsAnthony, Larry Cameron, Flint, Dennis, Suh, Wonchul, Ye, Rick W., Rothman, Steven Cary, Tomb, Jean-Francois
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Primary Examiner(s)Robinson, Hope
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Application NumberUS13/296,944Publication NumberTime in Patent Office273 DaysField of SearchNoneUS Class Current435/137CPC Class CodesC12N 9/88 Lyases (4.)C12P 7/16 ButanolsC12P 7/26 KetonesC12P 7/40 containing a carboxyl group...C12Y 402/01009 Dihydroxy-acid dehydratase ...Y02E 50/10 Biofuels, e.g. bio-diesel
