YEAST WITH INCREASED BUTANOL TOLERANCE INVOLVING A MULTIDRUG EFFLUX PUMP GENE

• US 20100221801A1
• Filed: 02/24/2010
• Published: 09/02/2010
• Est. Priority Date: 02/27/2009
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: VAN DYK, TINA K.
• Granted Patent: US 8,614,085 B2
• Time in Patent Office: Days
• US Class Current: 435/160
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Increasing tolerance to butanol in yeast has been accomplished by decreasing activity of Pdr5p encoded by an endogenous PDR5 gene. A deletion mutation of the PDR5 gene led to improved growth yield in the presence of butanol. Yeast cells with reduced Pdr5p activity, or other multidrug resistance ATP-binding cassette transporter protein activity encoded by CDR1 or BFR1, and a butanol biosynthetic pathway may be used for improved butanol production

Claims

1. A recombinant yeast cell comprising at least one genetic modification that reduces Pdr5p activity wherein said yeast cell has improved tolerance to butanol and isomers thereof as compared to a yeast cell that lacks said modification.

3. The recombinant yeast cell of claim 1 or 2, wherein said cell comprises a butanol biosynthetic pathway.

4. The recombinant yeast cell of claim 3 wherein the butanol biosynthetic pathway is selected from the group consisting of:

a) a 1-butanol biosynthetic pathway

b) a 2-butanol biosynthetic pathway; and

c) an isobutanol biosynthetic pathway.

5. The recombinant yeast cell of claim 4 wherein the 1-butanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions:

a) acetyl-CoA to acetoacetyl-CoA, as catalyzed by acetyl-CoA acetyltransferase;

b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed by 3-hydroxybutyryl-CoA dehydrogenase;

c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed by crotonase;

d) crotonyl-CoA to butyryl-CoA, as catalyzed by butyryl-CoA dehydrogenase;

e) butyryl-CoA to butyraldehyde, as catalyzed by butyraldehyde dehydrogenase; and

f) butyraldehyde to 1-butanol, as catalyzed by 1-butanol dehydrogenase.

6. The recombinant yeast cell of claim 4 wherein the 2-butanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions:

a) pyruvate to alpha-acetolactate, as catalyzed by acetolactate synthase;

b) alpha-acetolactate to acetoin, as catalyzed by acetolactate decarboxylase;

c) acetoin to 2,3-butanediol, as catalyzed by butanediol dehydrogenase;

d) 2,3-butanediol to 2-butanone, as catalyzed by butanediol dehydratase; and

e) 2-butanone to 2-butanol, as catalyzed by 2-butanol dehydrogenase.

7. The recombinant yeast cell of claim 4 wherein the isobutanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions:

a) pyruvate to acetolactate, as catalyzed by acetolactate synthase;

b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by acetohydroxy acid isomeroreductase;

c) 2,3-dihydroxyisovalerate to α

-ketoisovalerate, as catalyzed by acetohydroxy acid dehydratase or dihydroxyacid dehydratase;

d) α

-ketoisovalerate to isobutyraldehyde, as catalyzed by a branched-chain keto acid decarboxylase; and

e) isobutyraldehyde to isobutanol, as catalyzed by a branched-chain alcohol dehydrogenase.

8. The recombinant yeast cell of claim 1 or 2 wherein the cell has at least about a 1.8-fold improvement in growth yield in 0.75% (w/v) isobutanol as compared to a parental cell that does not have said genetic modification.

10. A method for the production of butanol or an isomer thereof comprising growing said yeast cell of claim 3 under conditions wherein butanol or an isomer thereof is produced and optionally recovering said butanol or isomer thereof.

11. The method of claim 10 wherein the butanol is isobutanol.

2. A recombinant yeast cell comprising at least one genetic modification that decreases activity of a multidrug resistance ATP-binding cassette transporter protein encoded by a PDR5, CDR1, or BFR1 gene wherein said yeast cell has improved tolerance to butanol and isomers thereof as compared to a yeast cell that lacks said modification.

9. The yeast cell of claim 2 wherein the genetic modification substantially eliminates expression of a multidrug resistance ATP-binding cassette transporter protein encoded by a PDR5, CDR1 or BFR1 gene that is endogenous to the cell.

12. A method for producing a recombinant yeast cell having increased tolerance to butanol comprising:

a) providing a recombinant yeast cell comprising a butanol biosynthetic pathway selected from the group consisting of;

i) a 1-butanol biosynthetic pathway

ii) a 2-butanol biosynthetic pathway; and

iii) an isobutanol biosynthetic pathway; and

b) engineering the yeast cell of (a) to comprise at least one genetic modification which decreases activity of a multidrug resistance ATP-binding cassette transporter protein encoded by a PDR5, CDR1 or BFR1 gene.